lignocellulose degradation and humus - helda -

Lignocellulose degradation and humus modification by

the fungus Paecilomyces inflatus

Beata Kluczek-Turpeinen

Division of MicrobiologyDepartment of Applied Chemistry and Microbiology

University of Helsinki

Academic Dissertation in Microbiology

To be presented with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in Auditorium at the Viikki Koetila

(Koetilantie 5) of the University of Helsinki on November the 9th 2007at 12 o´clock noon.

Helsinki 2007

vkirja_taitto_final_kielitarkastettu_pieni_fontti.indd 1 14.10.2007 23:01:37

Supervisor: Prof. Annele HatakkaDepartment of Applied Chemistry and MicrobiologyUniversity of Helsinki, Finland

Co-supervisor: Prof. Martin Hofrichter Chair of Environmental BiotechnologyInternational Graduate SchoolZittau, Germany

Reviewers: Doc. Merja ItävaaraVTT BiotechnologyEspoo, Finland

Dr. Petr BaldrianLab of Wood Rotting Fungi, Institute of MicrobiologyCzech Academy of Sciences,Prague, Czech Republic

Opponent: Assoc. prof. Paul Ander Swedish University of Agricultural Sciences Department of Forest Products/Wood Sciences, Uppsala, Sweden

Printed: Helsinki University Printing House 2007Layout: Isto Turpeinen

ISSN 1795-7079ISBN 978-952-10-4245-4 paperbackISBN 978-952-10-4246-1 pdf version, http://ethesis.helsinki.fi

e-mail: [email protected] photo: Microscopic picture of Paecilomyces variotti. (photo by David Ellis, Univer-

sity of Adelaide, Australia)


Table of Contents

List of original publications ...................................................................................................5The author´s contribution .................................................................................................5

Abbreviations ............................................................................................................................6Abstract .....................................................................................................................................7Tiivistelmä (Abstract in Finnish) ...........................................................................................81. INTRODUCTION .............................................................................................................9

1.1. Microfungi ...................................................................................................................91.1.1. Characteristics and importance of microfungi .......................................................... 91.1.2. Microfungi in compost ................................................................................................ 10

1.1.2.1. Composting environment................................................................................... 101.1.2.2. Occurrence and role of microfungi in compost............................................. 14

1.1.3. Paecilomyces inflatus .................................................................................................... 171.2. Lignocellulosic materials and their degradation .................................................. 21

1.2.1. Lignin .............................................................................................................................. 211.2.2. Lignin biodegradation .................................................................................................. 24

1.2.2.1. Lignin -degrading microorganisms .................................................................. 241.2.2.2. Lignin -degrading microfungi ........................................................................... 26

1.2.3. Lignin degrading enzymes ........................................................................................... 271.2.3.1. Characteristic of lignin-degrading enzymes .................................................... 271.3.2.2. Peroxidases............................................................................................................ 271.3.2.3. Laccase .................................................................................................................. 28

1.2.4. Cellulose and hemicellulose ........................................................................................ 311.2.5. Cellulose and hemicellulose biodegradation ............................................................ 31

1.3. Humic substances ................................................................................................... 341.3.1. Occurrence and formation of humic substance .................................................... 341.3.2. Biodegradation of humic substances ........................................................................ 35

2. AIMS OF THE STUDy ................................................................................................. 393. MATERIALS AND METHODS ................................................................................. 40

3.1. Compost samples ..................................................................................................... 403.2. Fungal strains ........................................................................................................... 403.3. Main experimental methods ................................................................................... 403.4. Additional methods.................................................................................................. 41

3.4.1. Determination of molecular mass distribution of compost lignocellulose (unpublished) .................................................................................................. 413.4.2. Conditions for laccase production in liquid cultures (unpublished) ..................... 42


4. RESULTS ........................................................................................................................... 434.1. Lignin degradation (I and IV) ................................................................................ 434.3. Lignin-degrading laccase ........................................................................................ 444.4. Cellulose and hemicellulose degradation .............................................................. 464.5. Cellulose-degrading endoglucanase (III and IV) ................................................. 494.6. Modification of humic substances ........................................................................ 49

5. DISCUSSION ................................................................................................................... 505.1. Degradation of lignin (I and IV) ........................................................................... 505.2. Lignin-degrading laccase ......................................................................................... 525.3. Degradation of cellulose and hemicellulose (IV) ................................................ 555.4. Cellulose-degrading endoglucanase (EG; III and IV) ....................................... 565.5. Modification of humic substances (II) ................................................................. 58

6. CONCLUSIONS AND FUTURE PERSPECTIVES ............................................. 607. ACKNOWLEDGEMENTS .......................................................................................... 628. REFERENCES .............................................................................................................. 63


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List of original publications

The thesis is based on the following publications referred to in the text by Roman nu-merals I- IV. In addition, unpublished data are also presented.

I Kluczek-Turpeinen B., Tuomela M., Hatakka A. and Hofrichter M. 2003 Lignin deg-radation in a compost environment by the deuteromycete Paecilomyces inflatus. Applied Microbiology and Biotechnology 61: 374-379

II Kluczek-Turpeinen B., Steffen K.T., Tuomela M., Hatakka A. and Hofrichter M. 2005 Modification of humic acids by the compost-dwelling deuteromycete Paecilomyces inflatus. Applied Microbiology and Biotechnology 66: 443-449

III Kluczek-Turpeinen B., Maijala P., Tuomela M., Hofrichter M. and Hatakka A. 2005 Endoglucanase activity of compost-dwelling fungus Paecilomyces inflatus is stimulated by humic acids and other low molecular mass aromatics. World Journal of Microbiology and Biotechnology 21: 1603-1609

IV Kluczek-Turpeinen B., Maijala P., Hofrichter M. and Hatakka A. 2007 Degradation and enzymatic activities of three Paecilomyces inflatus strains grown on diverse lignocellu-losic substrates. International Biodeterioration and Biodegradation 59: 283-291

The author´s contribution

I Beata Kluczek-Turpeinen planned the experiments, did the laboratory work. She inter-preted the results and wrote the paper.

II Beata Kluczek-Turpeinen planned the experiments, did the laboratory work except the HPSEC analyses. She interpreted the results and wrote the paper.

III Beata Kluczek-Turpeinen planned the experiments, did the laboratory work, ana-lyzed the data and wrote the paper together with Pekka Maijala.

IV Beata Kluczek-Turpeinen planned the experiments, did the laboratory work. She in-terpreted the results and wrote the paper. Pekka Maijala supervised part of the practical work and took part in the interpretation of the results and writing the paper.


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Abbreviations

ABTS 2,2`-azinobis(3-ethylbenzthiazoline-6-sulphonate)BGL β-glucosidasesCDH cellobiohydrolaseCM-cellulose carboxymethyl-cellulose DHP dehydrogenation polymer (synthetic lignin)EG endoglucanaseFA fulvic acidFPA filter paper assayG guaiacylGM composted grape marcHA humic acidHBT hydroxybenzotriazoleHPSEC high performance size exclusion chromatographyHS humic substancesITS internal transcribed spacer kDa kiloDaltonLiP lignin peroxidaseMnP manganese peroxidaseMSW municipal solid wasteMW molecular weightMM molecular masspI isoelectric pointRNA ribonucleic acidS syringylSHA soil humic acidS sewage sludgeSSC solid-state cultivationVP versatile peroxidasesWC wood compost


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Abstract

Composting is the biological conversion of solid organic waste into usable end prod-ucts such as fertilizers, substrates for mushroom production and biogas. Although com-posts are highly variable in their bulk composition, composting material is generally based on lignocellulose compounds derived from agricultural, forestry, fruit and veg-etable processing, household and municipal wastes. Lignocellulose is very recalcitrant; however it is rich and abundant source of carbon and energy. Therefore lignocellulose degradation is essential for maintaining the global carbon cycle. In compost, the active component involved in the biodegradation and conversion processes is the resident mi-crobial population, among which microfungi play a very important role. In composting pile the warm, humid, and aerobic environment provides the optimal conditions for their development. Microfungi use many carbon sources, including lignocellulosic poly-mers and can survive in extreme conditions. Typically microfungi are responsible for compost maturation.

In order to improve the composting process, more information is needed about the microbial degradation process. Better knowledge on the lignocellulose degradation by microfungi could be used to optimize the composting process. Thus, this thesis focused on lignocellulose and humic compounds degradation by a microfungus Paecilomyces in-flatus, which belongs to a flora of common microbial compost, soil and decaying plant remains. It is a very common species in Europe, North America and Asia. The lignocel-lulose and humic compounds degradation was studied using several methods including measurements of carbon release from 14C-labelled compounds, such as synthetic lignin (dehydrogenative polymer, DHP) and humic acids, as well as by determination of fibre composition using chemical detergents and sulphuric acid. Spectrophotometric enzyme assays were conducted to detect extracellular lignocellulose-degrading hydrolytic and oxidative enzymes.

Paecilomyces inflatus secreted clearly extracellular laccase to the culture media. Laccase was involved in the degradation process of lignin and humic acids. In compost P. infla-tus mineralised 6–10% of 14C-labelled DHP into carbon dioxide. About 15% of labelled DHP was converted into water-soluble compounds. Also humic acids were partly min-eralised and converted into water-soluble material, such as low-molecular mass fulvic acid-like compounds. Although laccase activity in aromatics-rich compost media clearly is connected with the degradation process of lignin and lignin-like compounds, it may preferentially effect the polymerisation and/or detoxification of such aromatic com-pounds. P. inflatus can degrade lignin and carbohydrates also while growing in straw and in wood. The cellulolytic enzyme system includes endoglucanase and β-glucosidase. In P. inflatus the secretion of these enzymes was stimulated by low-molecular-weight aromat-ics, such as soil humic acid and veratric acid. When strains of P. inflatus from different ecophysiological origins were compared, indications were found that specific adaptation strategies needed for lignocellulosics degradation may operate in P. inflatus. The degrada-tive features of these microfungi are on relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not es-tablished. The results of this study may help to understand, control and better design the process of plant polymer conversion in compost environment,with a special emphasis on the role of ubiquitous microfungi.


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Tiivistelmä (Abstract in Finnish)

Kompostoitumisella tarkoitetaan kiinteän orgaanisen aineen biologista muuntumista hyödynnettäviksi lopputuotteiksi, kuten vaikkapa lannoitteiksi, ruokasienten kasvatus-alustoiksi sekä biokaasuksi. Kompostien koostumus vaihtelee suuresti; kuitenkin useim-miten kompostin perustana ovat kasviperäiset materiaalit sekä talousjätteet. Kasviaines on pääosin rakentunut puuaineesta eli ligniinistä sekä selluloosasta. Kasviainekseen on sitoutunut valtava määrä hiiltä ja energiaa. Kokonaisuutena lignoselluloosan hajotus on välttämätöntä maapallon hiilen kierron kannalta.

Kompostissa kompostin mikrobit, erityisesti mikrosienet, muuntavat ja hajottavat lignoselluloosaa. Kompostin lämmin, kostea ilma takaa mikrosienille ihanteelliset olot sienten kasvuun ja kasviaineksen hajotukseen. Monet mikrosienet ovat sopeutuneet selviytymään äärimmäisissäkin olosuhteissa.

Kasviaineksen kompostoitumistehokkuutta voidaan lisätä, jos kompostoitumisen mikrobiologia ja erityisesti lignoselluloosan hajotus tunnetaan hyvin. Tämän väitöskir-jatyön tarkoituksena oli tutkia kompostissa elävän Paecilomyces inflatus –mikrosienen lignoselluloosan sekä humusyhdisteiden hajotusta. Sieni on sangen tavallinen maaperän ja kompostien sieni sekä Euroopassa, Aasiassa että Pohjois-Amerikassa. Sienen on to-dettu kykenevän kasvamaan myös puussa. Lignoselluloosan ja humusyhdisteiden hajo-tusta tutkittiin usein eri menetelmin, mm. käyttämällä radioaktiivisella hiilellä leimattuja malliyhdisteitä, analysoimalla kasviaineksen puuaineen määrää sekä tutkimalla sienen pu-uainetta hajottavien entsyymien erittymistä kasvualustaan.

Tutkimuksissa selvisi että kompostista eristetty mikrosieni P. inflatus osoitti selvästi solunulkoisten hapettavien entsyymien kuten lakkaasin tuottoa. Lakkaasin avulla sieni pystyi hajottamaan jonkin verran puuainetta ja humusyhdisteitä, kuten humushappoja, joiden tärkein lähtöaine on puuaine eli ligniini.

Kompostissa mikrosieni hajotti 6–10% radioaktiivisella hiilellä leimatusta puuainees-ta hiilidioksidiksi. Leimautuneita vesiliukoisia yhdisteitä muodostui 15%. Sieni tuotti humushaposta hiilidioksidia ja pienimolekyylisiä yhdisteitä kompostiviljemässä. Paitsi kompostissa P. inflatus hajottaa puuainetta ja hiilihydraatteja myös olkialustalla ja puussa. Sieni näyttää olevan hyvin sopeutunut myös näihin olosuhteisiin. Selluloosaa hajottavista entsyymeistä sieni tuotti endoglukanaasia ja β-glukosidaasia.

Tulokset osoittavat, että näillä sienillä voi olla merkittävä osuus ligniinin, humuksen ja lignoselluloosan hajotuksessa niin kompostissa kuin maassakin, eli ympäristöissä, missä varsinaisten puuta lahottavien kantasienten elinkyky on rajoittunut. Tutkimustyön tu-lokset auttavat paremmin ymmärtämään, hallitsemaan ja suunnittelemaan kasviaineksen kompostihajotusta.

Tulokset vahvistavat mikrosienten keskeistä osuutta osana hyvän kompostin toimintaa.


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1. INTRODUCTION

1.1. Microfungi

1.1.1. Characteristics and importance of microfungi

Microfungi are diverse group of fungi consisting of yeasts and molds (Gravesen et al. 1994). From the taxonomic point of view, most microfungi belong to the Ascomycetes, mitosporic fungi (Deuteromycetes) and Zygomycetes. About 29 000 ascomycetous spe-cies are known so far. Mitosporic fungi, formerly designated as Deuteromycetes, account for about 17 000 species. The Zygomycetes are unimpressive in numbers of species, ap-proximately only 700 being known (Gow and Gadd 1996). The fungal body consists of microscopic threads called hyphae, extending through the substrate through which they grow. Typically only the “fruiting body” of the fungus is visible, producing thousands of tiny spores that are carried by the air, spreading the fungus to new locations. Spores are produced in a variety of ways and occur in a bewildering array of shapes and sizes. In spite of this diversity, spores are quite constant in their shapes, sizes (about 2–20 µm), colour and form. Thus these characteristics are very useful for identification of micro-fungi. The most basic difference between spores lies in their method of initiation, which can be either sexual or asexual (Carlile et al. 2001).

Microfungi are well adapted to extreme environmental conditions. They tolerate a wide range of temperature, pH, dryness, oxygen concentrations and ultraviolet radiation better than the wood-rotting basidiomycetes called white or brown rot fungi. In addi-tion they are found in all climatic zones ranging from the poles to the tropics (Blanchette 2000, Blanchette et al. 2004). Generally, fungi prefer an acidic environment (Deacon 1997) although microfungal activities occur within a board pH range of between 3.7 and 8.6 (Daniel and Nilsson 1998). Microfungi are found in acid coniferous forest soils in addition to neutral soils and composts (Tuomela et al. 2000, Daniel and Nilsson 1998) and they can successfully colonize exposed aerial surfaces, in conditions, which may be preventative to the growth of other microorganisms (Carlile et al. 2001, Blanchette 2000). Moreover, microfungi can protect themselves by relatively quick growth in natu-ral niches and by the production of antibiotics and toxic substances (mycotoxins). They can also serve as feed for insects and as symbiotic partners with algae and cyanobacteria in lichens (Gravesen et al. 1994).

Microfungi are common saprophytes that exist in: soil Trichoderma, Penicillium; (Dom-sch et al.1980), compost Chaetomium (Chefetz et al. 1998), wood Xylaria and Hypoxylon (Pointing et al. 2003), and in water environment Ophioceras dolichostomum, Savoryella lignicola (Bucher et al. 2004). Many microfungi are important plant pathogens, including Ophios-toma novo-ulmi in Dutch elm disease (Gow and Gadd 1996), Claviceps purpurea causing er-got of cereals (Gravesen et al. 1994)) and Fusarium solani f. sp. glycines that cause root rots of soybean (Lozoyova et al. 2006). Some other microfungi are parasities of insects e.g. Beauveria or nematodes e.g. Arthrobotrys (Gow and Gadd 1996).


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In soil, microfungi usually exist in the organic upper layers (humus and topsoil), al-though some species have also been found in underlying rocky layers (subsoil) (Dix and Webster 1995). Similarly in compost they mainly occupy the upper parts of compost (>10 cm depth) where oxygen remains available (Millner et al. 1977). In both habitats microfungi together with bacteria, other fungi and animals participate in the decomposi-tion of organic matter to carbon dioxide and humus (Dix and Webster 1995).

Microfungi are able to colonize and cause soft-rot decay of wood (Daniel and Nils-son 1998). Soft rot decomposition can even occur in wood with high content of tannins and other compounds normally resistant to microbial attack. In addition soft rot is asso-ciated with hot, wet and cold conditions, which inhibits colonization by the more aggres-sive white and brown rotting fungi (Blanchette 2000, Blanchette et al. 2004). The wood decayed by soft rot has a brown soft appearance that is cracked and checked when dry (Blanchette 1995). Microfungi preferably colonize and degrade hardwood. In softwood the rate of the wood decay by microfungi is generally lower than in hardwood (Kuhad et al. 1997). Weight losses of up to 50 % in birch wood (hardwood) and 20 % in pine wood (softwood) within 3 months caused by these fungi have been reported (Nilsson et al. 1989, Ferraz and Duran 1995). Microfungi preferentially metabolize wood polysac-charides and produce an array of cellulolytic and hemicellulolytic activities that may con-tribute to the degradation of plant cell wall material (Kubicek and Penttilä 1998, de Vries and Visser 2001, Tribak et al. 2002). They are also capable of some direct transformation of lignin from the outer layers of the cell walls, however they leave the middle lamella in-tact (Blanchette 1995). On the other hand , many microfungi have been reported to de-grade synthetic lignin to CO2 and water-soluble products (Haider and Trojanowski 1975, Rodriguez et al. 1996b, Regalado et al. 1997, Gonzalez et al. 2002, Liers et al. 2006) and rapidly convert lignin-related phenolic compounds (Ander et al. 1984, Betts and Dart 1988, Bugos et al. 1988, Hofrichter et al. 1993, Hofrichter et al. 1994, Leitão et al. 2007). Such abilities of microfungi may be linked to their capability for lignin degradation. Ta-ble 1 presents the spectrum of microfungi species involved in the production of various lignocellulolytic enzymes in solid state cultivation systems.

In addition to the important role of microfungi in carbon cycling, they are also in-volved in many biotechnological processes. These processes include: brewing, winemak-ing, baking, cheese making and the preparation of other fermented food (e.g. tempe, miso, angkak, soy sauce) together with edible mushroom production are the most im-portant microfungal applications. Production of enzymes (amylase, cellulase, invertase, lipase, pectinase, proteinase, rennin and xylanase), organic acids (citric, itaconic and lac-tic acids), antibiotics and other pharmaceuticals (penicillin, mevinolin, cephalosporin, griseofulvin and cyclosporine) by fungi are common processes that have been reviewed (Bennet 1998 and Demain 1999).

1.1.2. Microfungi in compost

1.1.2.1. Composting environment

The degradation of organic wastes is a natural process and begins almost as soon as the wastes are generated. Composting is a means of controlling and accelerating the decom-position process. This involves the self-heating and aerobic biological breakdown of or-


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Substrate Microfungus Enzyme ReferenceWheat bran, wheat straw, banana leaf waste

Aspergillus sp., A. ter-reus, A. niger

CM-cellulase, FPA, β-glucosidase, CBH, xylanase, laccase, LiP

Ghanem et al. 2000, Hanif et al. 2004,Shah et al. 2005

Softwood Kraft lignin

Botryosphaeria sp. laccase Dekker et al. 2001

Wheat straw Botrytis cinerea cellulase, xylanase Thygesen et al. 2003

Wheat bran, sugar beet pulp, wheat straw, palm fruit fibre

Chaetomium globosum xylanase, cellulases Wiacek-Zychlinska et al. 1994, Umikalsom et al. 1998

Wheat straw, com-post

Chaetomium thermophilum xylanase, laccase Latif et al. 2006, Che-fetz et al. 1998,

Bagasse Humicola grisea var. ther-moidea

CBH, FPA, β-glucosidase, xyla-nase

De-Paula et al. 1999, Salles et al. 2005

Bagasse, wheat straw, rice straw, rice husks, barley bran

Melanocarpus sp., M. albomyces

xylanases, endogluca-nase, CBH

Prahbu and Meheshwari 1999, Saraswat and Bisaria 2000, Jatinder et al. 2006, Kaur et al. 2006

Bagasse, corn cob, rice straw, wheat straw, wheat bran

Myceliophthora sp. cellulases, xylanases Badhan et al. 2007

Wheat straw Neurospora crassa endoglucanase Romero et al. 1999Wheat straw Paecilomyces thermophila xylanase yang et al. 2006

Rice straw, Corn cob, oat husk, agri-cultural residues

Penicillium sp., P. simpli-cissimum, P. janthinellum, P. brasilianum

laccase, cellulasesxylanases

Rahman et al. 2003,Thygesen et al. 2003, Oliveira et al. 2006Zeng et al. 2006

Table 1. Microfungal species used for the production of various lignocellulolytic enzymes in solid state cultivation systems


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Bagasse, grass clip-pings, rice straw

Scytalidium thermophilum endoglucanase, exo-glucanase, β-glucosidase,

Ögel et al. 2001, Kaur et al. 2006

Wheat straw Sporotrichum thermophile xylanase Topakas et al. 2003

Wheat straw, wheat bran, baggase, agri-cultural residues

Thermoascus auranticus endoglucanase, xylanase, phenol oxi-dase

Machuca et al. 1998, Kalogeris et al. 2003, Milagres et al. 2003

Corncob Thermomyces lanuginosus xylanase Damaso et al. 2000

Wheat straw Trichoderma longibrachia-tum

CM-cellulase, β- glucosidase, laccase, MnP

Velazquez -Cedeňo et al. 2004

Birchwood Xylaria polymorpha* endoglucanase, β-glucosidase, estera-se, xylanase, laccase

Liers et al. 2006

ganic materials, with successions of different microbes, in which temperature, pH and availability of nutrients constantly change (Biddlestone and Gray 1985, Epstain 1997, Tuomela et al. 2000, Ryckeboer et al. 2003b). A scheme of composting process in shown in Figure 1. Composting may mineralize the simpler and more easily assimilated com-pounds and humify complex substrates into usable end products such as fertilizers, sub-strates for mushroom production and biogas (Crawford 1983, Epstain 1997). Although composts are highly variable in bulk composition, they are generally based on lignocellu-lose compounds, together with other substrates derived from agricultural, forestry, fruit and vegetable processing as well as household and municipal wastes.

Successful composting depends on a number of optimal factors including : an ade-quate supply of oxygen, correct particle size, moisture, C/N ratio and pH. These factors influence the type of microorganisms, species diversity and the rate of decomposition (Crawford 1983). The key parameters of composting are given in Table 2. The complex-ity of degraded plant materials and the quality of the final product may depend upon the type of waste (Biddlestone and Gray 1985).

The resident microbial community in compost consists of bacteria, actinomycetes and fungi, Resident microbial communities have recently been reviewed by Tuomela et al. (2000). During the various composting phases different microbial communities pre-dominate, each of which is adapted to the particular environment (Ryckeboer et al. 2003 a, b Table 3). At the beginning of composting mesophilic bacteria predominate, but when the temperature increases to over 40°C, thermophilic bacteria and fungi predomi-nate in the compost. Temperatures of over 60°C are critical for microorganisms thus microbial activity decreases dramatically but after the compost has cooled mesophilic bacteria and actinomycetes again predominate (Ryckeboer et al. 2003 b). Among micro-


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bial organisms, microfungi play a very important role. They can use many carbon sourc-es including lignocellulose polymers and they can survive in variable conditions. There-fore, microfungi are mainly responsible for compost maturation (Maheshwari et al. 2000, Tuomela et al. 2001) and compost seems to be an excellent habitat for them since it con-tains all organic substrates necessary for microbial growth and reproduction.

During the composting process temperature, pH and nutrient availability constantly change therefore these factors influence the types of microorganisms, species diversity and the rate of metabolic activities. Degradation of waste materials in compost proceeds in three phases: (i) the mesophilic phase, (ii) the thermophilic phase and (iii) the cooling and maturation phase, all of which differ in temperature, pH values and microbial con-sortia (Figure 2).

Figure 1. The compost-ing process and impor-tant compost factors affecting this process adapted from Itävaara et al. (1995)

Major parameters Optimum valueNutrient balance (C:N ratio) 35:1

Water content 50–75 % depends on material

Particle size 12.5 mm for agitated plants and forced aeration50 mm for windrows and natural aeration

Air flow0.6-1.8 m3 air d-1 kg-1 volatile solids during thermophi-lic phase, being progressively decreased during cooling down and maturing

pH 6.5–8.0

Oxygen concentration > 10 %

Temperature 55 °Cspan 50–65°C

Table 2. Composting parameters (Biddlestone and Gray 1985)


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Table 3. Species diversity of the dominant microorganisms isolated during different com-posting phases (van Heerden et al. 2002 and Ryckeboer et al. 2003a).

Composting phase Prokaryotes Fungi

Mesophilic phase

Bacillus maceransStaphylococcus saprophiticusFlavobacterium sp.Streptomyces sp.Rhodococcus rhodochrousMicrococcusNocardia otitidiscaviarium

Aspergillus flavusAspergillus nigerAspergillus ustusPenicillium sublateritumEupenicillium cinnamopurpureumCladosporium cladosporioides

Thermophilic phase

Enterobacter cloacaeCoryneform sp.Paenibacillus maceransBacillus licheniformisStaphylococcus capitisBrevibacillus agri

Absidia corymbiferaPenicillium diversumPaecilomyces variotiiRhizomucor pusillusThermomyces lanuginosusThermomyces. ibananensis

Cooling and maturation phase

Alcaligenes denitrificansProteus vulgarisPseudomonas aeruginosaSerratia marcescensCellulomonas cellulansBacillus sphaericusFlavobacterium mizutaii

Fusarium solaniPaecilomyces lilacinusCoprinus lagopusMucor sp.Thrichothecium sp.Geotrichum candidumMemnoniella echinata

1.1.2.2. Occurrence and role of microfungi in compost

Microfungi are the main components of the microflora that develop in heaped masses of plant material and piles of agricultural and forestry products wherein a warm, humid and aerobic environment provides the best conditions for their development. They grow in compost in all phases but may disappear temporarily during peak heating (Trambira-jah et al. 1995). Microfungi constitute a heterogeneous physiological group of various genera in the Ascomycetes, Zygomycetes and mitosporic fungi formarly know as Deu-teromycetes (Maheshwari et al. 2000).

Van Heerden et al. (2002) and Ryckeboer et al. (2003a) followed the succession of microfungi in a compost environment. They found a freshly made compost heap con-tains a variety of soil and leaf-inhabiting fungal genera. The majority of these fungi are mesophiles with maximum growth temperatures between 25 and 30°C whereas other genera are thermotolerant and are capable of growth over the range of 40 to 50°C. At temperature above 60°C is the upper limit of growth for fungi in addition to all other


15

eukaryotes (Kane and Mullins 1973). In some studies the raw material of compost has been found to contain approximately 106 microbial counts of mesophilic fungi per gram of raw material and Aspergillus and Penicillium are the predominant fungal genera (von Klopotek 1962, Trambirajah et al. 1995, van Heerden et al. 2002). As the pile tempera-ture increases to the thermophilic range, thenumber of fungi rises and they efficiently inhabit the pile. However, the counts of the fungi decreases to 103 per g of compost as the temperature rises to above 60°C and at 64°C all these fungi disappear (von Klo-potek 1962, Trambirajah et al. 1995). Interestingly, the mesophilic fungus Cladosporium cladosporioides was able to grow over the 64 to 65°C range (von Klopotek 1962). The fungi survive at high temperatures which is most likely due to the short duration of the exposure to the high temperatures (Trambirajah et al. 1995). Temperature tolerance also differs within genera and even within the fungal species. The growth substrate may also have some influence on temperature tolerance (Ofosu-Asiedu and Smith 1973a). Sporo-trichum thermophile Apinis (syn. Myceliophthora thermophila) produces extracellular cellulases on sugarcane bagasse even at 65°C (El-Naghy et al. 1991), whereas Talaromyces emersonii is still active and can grow after four weeks at elevated temperatures (Ofosu-Asiedu and Smith 1973a). As the temperature in compost falls below 60°C, both mesophilic and thermophilic microfungi start to re-colonise the substrate (von Klopotek 1962, Tram-birajah et al. 1995). Among the mesophilic fungi a few lignin-degrading Basidiomycota including Coprinus sp., Panaeolus sp., Corticium coronilla, Trametes sp. and Phanerochaete sp. have been isolated from compost at the cooling and maturation phases or from mature compost (von Klopotek 1962, Granit et al. 2007). The basidiomycete Coprinus cinereus is an example of a degrader of polymers, otherwise resistant to degradation. This fungus has a maximum growth temperature of about 40°C and prefers an alkaline environment (Dix and Webster 1995, Deacon 1997).

Figure 2. Composting phases modified from Biddlestone and Gray (1985)


16

Most microfungi are obligate aerobes consequently they have a lower tolerance for low oxygen partial pressure than bacteria. For this reason, microfungi mostly live in the outer layer of compost where they grow both as unseen filaments and fuzzy grey or white colonies on the compost surface. Thus, oxygen can be a limiting factor not only for the growth but also for their metabolic activity (Walsh 1972). Interestingly, oxygen deficiency brings about morphogenetic changes in Talaromyces (Penicillium) duponti (Coon-ey and Emerson 1964). The fungus forms only a conidial stage (Penicillium) in aerobic cultures. The sexual stage (Talaromyces) is initiated in agar cultures only when they are flushed with nitrogen.

Compost microfungi are less sensitive to environments with low moisture and pH than bacteria. Therefore microfungi can attack organic residues that are too dry and acidic or too low in nitrogen for bacterial decomposition. Nitrogen addition often in-creases the rate of lignin attack by most microfungi, in contrast to that found for the basidiomycetous white rotting fungi (Daniel and Nilsson 1998). Nitrogen availability is often also the limiting factor for cellulose degradation (Dix and Webster 1995).

Both thermophile and mesophile microfungi are responsible for the decomposition of many complex plant polymers during composting. They break down otherwise re-calcitrant debris, enabling bacteria to continue further the decomposition process after most of the cellulose has been exhausted. A range of cellulolytic microfungi colonize after peak-heating, then grow over the next 10 to 20 days. They rapidly decompose cel-lulose in compost, but enzyme activity of the respective culture filtrates has been found to be low when compared to that of the mesophilic fungus Hypocrea jecorina (anamorph Trichoderma reesei) (Bhat and Maheshwari et al. 1987). Interestingly, some compost fungi are unable to utilize cellulose for example Thermomyces lanuginosus, but this fungi can grow commensally by utilizing sugars generated by other fungi and perhaps also by using their mycelial breakdown products (Puchart et al. 1999). Moreover, several noncellulolytic spe-cies readily utilize xylan, the major hemicellulose component of the cell walls of many plants (Prabhu and Maheshwari 1999). The ability of microfungi to hydrolyze hemicellu-lose is probably more common than cellulose cleavage (Dix and Webster 1995). During the prolonged phase of warm temperature that follows peak-heating, a compost can lose up to 50 % of its dry weight. This loss comprises nearly two-thirds of the main plant cell wall components including cellulose and hemicellulose (Deacon 1997).

Lignin biodegradation is an important activity during composting because of its in-volvement in humification and release of nutrients to microorganisms. Tuomela et al. (2001) found a rather high mineralization of 14C-labelled synthetic lignin (14C-DHP) preparation by mixed microbial population in a compost environment. A noticeably higher degradation occurred at 35˚C and 50˚C (23–24 % ) than at 58˚C (7 % ). This points to an involvement of eukaryotic organisms (very probably microfungi) in the degradation process, since their activity is strongly suppressed at temperatures at 58ºC and above. Waksman et al. (1939) examined the lignin degradation capacity of some mi-croorganisms isolated from compost, and found that the thermophilic ascomycete Ther-momyces lanuginosus degraded 4.2 % of lignin at 50°C over 42 days. Thermoascus aurantia-cus degraded 15 % of wood lignin in a 21 day cultivation period (Machuca and Duran 1993). Aspergillus spp. has a high lignin-degrading capacity (Shah et al. 2005). Some of them have been isolated from compost (von Klopotek 1962, Van Heerden et al. 2002).


17

Low lignin-degrading activities have also been found for Paecilomyces spp., Thielavia ter-restris and Talaromyces thermophilus (Eslyn et al. 1975, Dix and Webster 1995). Melanocarpus albomyces a thermophilic ascomycete that commonly grows in the hottest parts of the compost (Prahbu and Maheshwari 1999) produces laccase that is able to bind to cellu-lose (Kiiskinen et al. 2004). Compost microfungi, which are known to have lignocellulo-lytic activity or which grow on lignocellulose or compost, are listed in Table 4.

1.1.3. Paecilomyces inflatus

The genus Paecilomyces includes 31 species divided into two sections Paecilomyces and Isari-oidea (Samson 1974). The classification is based on their morphological characteristics. The section Paecilomyces contains members that are often thermophilic whereas Isarioidea contains mesophiles, including several entomopathogenic or nematophagous species. However, data obtained from molecular studies modify the systematics of these fungi. Comparative analyses of ribosomal RNA gene sequences and internal transcribed spac-er (ITS) sequences indicate the polyphyletic character of the genus Paecilomyces (Obornik et al. 2001, Inglis and Tigano 2006). Paecilomyces is polyphyletic across three ascomycete orders, the Eurotiales, the Hypocreales and the Sordariales (Luangsa-ard et al. 2004, In-glis and Tigano 2006). The type species, Paecilomyces variotii, and thermophilic relatives

from the section Paecilomyces belong to the order Eurotiales (Trichocomaceae), whereas mes-ophilic species of the section Isarioidea and related to Paecilomyces farinosus are in the order Hypocreales (Clavicipitaceae and Hypocreaceae). In the Eurotiales anamorph Paecilomyces spe-cies are related to the teleomorphs Talaromyces and Thermoascus (Inglis and Tigano 2006). Only one species, Paecilomyces inflatus, has affinity for the order Sordariales (Luangsa-ard et al. 2004). Within the order Sordariales, P. inflatus is found to be associated with two ascomycetes, namely Chaetomium globosum and Neurospora crassa (Luangsa-ard et al. 2004).

Paecilomyces spp. are found in a great range of habitats, substrates and materials, in-cluding soils, litter, compost, sewage sludge, lakes, mouldy grain, straw and wood (Sam-son 1974, Domsch 1980, Harney and Widden 1990, Polishbook et al. 1996, del Rio et al. 2001, Ryckeboer et al. 2003b). These fungi prefer aerated habitats for growth and re-production, but are also capable of surviving in the anaerobic mullet gut (Walsh 1972, Mountfort and Rhodes 1991). As a saprophyte Paecilomyces spp. normally obtain nutrients from decaying organic matter, but they can also derive nutrients from living cells of in-sects as a parasite (Siddiqui and Mahmood 1996). Paecilomyces spp. can readily grow and reproduce over a wide temperature range from 5 to 55°C (Samson 1974, Pitt and Hock-ing 1999, Maheshwari et al. 2000, van Heerden et al. 2002).

The genus Paecilomyces has received only a little attention in lignocellulose degradation studies, despite the abundance of these fungi in agricultural wastes at different stages of decomposition (Tuomela et al. 2000). Although there are a few studies dealing with the degradation of lignocellulose components by these fungi, the results are partially con-tradictory and their enzymatic mechanisms are hardly understood (Kapoor et al. 1978, Kainsa et al. 1979, Mishra et al. 1979, Ghanen 1991, del Rio et al. 2001, Martinez et al. 2005). P. variotii efficiently degrades cellulose and lignin in wheat straw causing an in-crease in humus-like substances (Mishra et al. 1979). The ability to degrade cellulose and lignin in wood by Paecilomyces sp. has been discussed by Eslyn et al. (1975). These authors observed that the fungus depleted lignin more rapidly then other cell wall components.


18

Fung

usSu

bdiv

isio

n*S

ourc

e m

ater

ial

Deg

rade

d co

mpo

und

Ref

eren

ce

Lign

inC

ellu

lose

Hem

icel

lulo

seW

ood

Asp

ergill

usfu

miga

tus

Asc

omyc

otin

aW

C, H

C+

++

++

++

Flan

niga

n an

d Sa

goo

1977

, de

Vrie

s an

d V

isser

200

1

Chae

tomiu

m th

ermop

hilu

mva

r. cop

roph

ileva

r. dis

setum

Asc

omyc

otin

aM

SW, M

C, W

Cla

cca

++

++

+C

hefe

tz e

t al.

1998

, Hak

ulin

en e

t al.

2003

, Li

et a

l. 20

03

Eme

ricell

a ni

dulan

s (an

a-mo

rph:

Asp

ergill

us n

idu-

lans )

Asc

omyc

otin

aM

C,SC

, GC

-+

++

+(a

rabi

noxy

lan)

-Fe

rnan

dez-

Esp

inar

et a

l. 19

94, C

hi-

kam

atsu

et a

l. 19

99

Fusa

rium

oxysp

orum

Asc

omyc

otin

aG

C

++

++

++

Falc

on e

t al.

1995

, Rod

rigue

z et

al.

1996

b,A

bdel

-Sat

er a

nd E

l-Sai

d 20

01

Fusa

rium

solan

i A

scom

ycot

ina

MC,

GC

++

++

++

+Ro

drig

uez

et a

l. 19

96b,

Gop

inat

h et

al

. 200

5,Lo

zova

ya e

t al.

2006

Hum

icola

grisea

var.

ther-

moide

a D

eute

rom

ycot

ina

MSW

, WC,

HM

nd+

++

++

++

De-

Paul

a et

al.

1999

,Po

cas-

Fone

sca

et a

l. 20

00, S

alle

s et

al. 2

005

Tab

le 4

. D

egra

dat

ive

act

ivitie

s of m

icro

fungi is

ola

ted f

rom

com

post

for

diffe

rent

lignoce

llulo

se c

om

ponen

ts


19

Malb

ranc

hea

cinna

mone

a (=

Malb

rach

nea

pulch

ella

= T

hero

moidi

um su

lfereu

m)

Deu

tero

myc

otin

aM

C, H

C+

++

++

+

ndJa

in e

t al.1

979,

Mat

suo

and

yasu

i 19

85,

Mela

noca

rpus

albo

myces

(= M

yrioco

ccum

albom

yces)

Asc

omyc

otin

aM

Cla

cca

++

++

+nd

Prah

bu a

nd M

ahes

hwar

i 199

9, S

a-ra

swat

and

Bisa

ria 2

000,

Kiis

kine

n et

al.

2002

Mie

ttine

n-O

inon

en e

t al.

2004

M

ycelio

phth

ora

therm

ophi

la (=

Spo

rotri

chum

therm

o-ph

ile )

Asc

omyc

otin

aM

C, G

W

nd+

++

++

ndO

fosu

-Asie

du a

nd S

mith

197

3b,

Bhat

and

Mah

esw

ari 1

987,

El-N

aghy

et

al.

1991

,To

paka

s et a

l. 20

03

Paeci

lomyce

s sp.

Deu

tero

myc

otin

aM

SW, M

C,

HM

, G

C

++

++

+

(xyl

an)

+E

slyn

et a

l. 19

75, O

kolo

et a

l. 19

98,

del R

io e

t al.

2001

, Mar

tinez

et a

l. 20

05

Paeci

lomyce

s var

iotii

Deu

tero

myc

otin

aM

C+

++

++

+ (x

ylan

)+

Kel

ly e

t al.

1989

, Dix

and

Web

ster

19

95, G

opin

ath

et a

l. 20

05

Peni

cilliu

m ch

rysog

enum

(= P

enici

llium

nota

tum)

Asc

omyc

otin

aG

C

++

+

(xyl

an)

ndRo

drig

uez

et a

l. 19

94, F

alco

n et

al.

1995

, Rod

rigue

z et

al.

1996

a

Preu

ssia

fleisc

hhak

iiD

eute

rom

ycot

ina

GC

+nd

nd+

Hai

der a

nd T

roja

now

ski 1

975

Rhiz

omuc

or p

ussil

us(=

Muc

or p

usill

us)

Zyg

omyc

otin

aM

C, H

Cnd

+/-

+nd

Dix

and

Web

ster

199

5,Ra

hman

et a

l. 20

01


20

Scyta

lidiu

m th

ermop

hilu

m( =

Toru

la th

ermop

hila

)D

eute

rom

ycot

ina

MC

+

++

+nd

+Ja

in e

t al.

1979

, Öge

l et a

l. 20

01,

Öge

l et a

l. 20

06

Talar

omyce

s eme

rsoni

iA

scom

ycot

ina

MC,

SC,

WC,

G

C+

++

++

+O

fosu

-Asie

du a

nd S

mith

197

3b,

Tuoh

y et

al 1

993,

Mur

rey

et a

l. 20

01

Talar

omyce

s the

rmop

hilu

sA

scom

ycot

ina

GC,

MC,

HC

++

++

Dix

and

Web

ster

199

5

Thiel

avia

terr

estris

(=

Alle

scheri

a ter

restri

s)A

scom

ycot

ina

MC,

GC

++

+nd

+O

fosu

-Asie

du a

nd S

mith

197

3b, E

s-ly

n et

al.

1975

, Gilb

ert e

t al.

1993

Thero

moas

cus a

uran

tiacu

s A

scom

ycot

ina

MSW

++

++

++

+(+

)M

achu

ca a

nd D

uran

199

3, K

alog

eris

et a

l. 20

03, M

ilagr

es e

t al.

2003

Therm

omyce

s lan

ugin

osus

(= H

umico

la la

nugin

osa)

Deu

tero

myc

otin

aM

SW, S

C, G

C+

+/-

++

+

(xyl

an a

nd a

rabi

-no

xyla

n)

ndW

aksm

an e

t al.

1939

, Jai

n et

al.

1979

, Pu

char

t et a

l. 19

99, D

amas

o et

al.

2000

Trich

oderm

a ko

ning

iiD

eute

rom

ycot

ina

MSW

, GM

++

++

++

+

(xyl

an a

nd a

rabi

-no

xyla

n)

+G

erbe

r et a

l. 19

97, L

opez

et a

l. 20

06

* So

urce

mat

eria

l of

com

post

: MSW

= m

unic

ipal

solid

was

te, M

C =

mus

hroo

m c

ompo

st, S

C =

stra

w c

ompo

st, H

M =

man

ure,

hors

e co

m-

post

, WC

= w

ood

com

post

, HC

= h

ay c

ompo

st, G

C =

gar

den

com

post

, FC

= fo

od c

ompo

st, G

M =

gra

pe m

arc

a lig

nin

degr

adat

ion

not d

eter

min

ed, b

ut fo

und

to p

rodu

ce la

ccas

e, ph

enol

oxi

dase

nd =

not

det

erm

ined


21

About 50 % of lignin loss caused by P. variotii in beech sawdust has been reported, and the rate of lignin degradation dependes on culture conditions and the composition of fermentation medium (Ghanem 1991). In contrast, recent studies investigating hard-wood decay inoculated with Paecilomyces sp. using analytical pyrolysis - GC/MS revealed an increase in lignin proportion due to preferential removal of polysaccharides (del Rio et al. 2001, Martinez et al. 2005). In studies by Calvo et al. (1995), effluent from the pa-per industry treated with P. variotii had a very high alkali lignin loss (78 % ). However, a significant proportion of effluent alkali lignin was found to be attached to the fun-gal biomass. Lignin-related compounds such ferulic, syringic and p-coumaric acids and other phenols, are rapidly degraded by Paecilomyces spp. (Rauhoti et al. 1989, Ghosh et al. 2006, Mukherjee et al. 2006, Sachan et al. 2006). Recently Paecilomyces lilacinus was found to transform and even completely mineralize biphenyl (Gesell et al. 2001) and dibenzo-furan (Gesell et al. 2004). In addition P. variotii is able to utilize toluene as the sole carbon source and degrade it to CO2 (Estevez et al. 2005).

Most of Paecilomyces spp. produce a range of glucanases that hydrolyze hemicelluloses and celluloses (Kelly et al. 1989, Almeida e Silva et al. 1995, Okolo et al. 1998, Tribak et al. 2002, Yang et al. 2006). Some of these enzymes have been purified and characterized (Kelly et al. 1989, Okolo et al. 1998). Paecilomyces farinosus (Fakoussa and Frost 1999), Pae-cilomyces sp. (Donnison et al. 2000) and P. variotii (Rahouti et al.1989) also secrete laccase-type phenol oxidases when they grow on phenolic compounds.

P. variotii has been utilized in the industrial process for the production of microbial protein. In this process known as “Pekilo”, the fungus is grown on a variety of ligno-cellulosic wastes, such as wood hydrolyzates, spent sulphite liquor, molasses and vinasse (Romantschuk and Lehtomäki 1987). The resulting protein produced contains all the es-sential amino acids for animal feed.

1.2. Lignocellulosic materials and their degradation

In nature, lignocellulose containing biomass is the major source of renewable organic matter produced by plant photosynthesis. Lignocellulosic wastes are formed in plant pro-duction (agriculture and forestry) and industrial processes (pulp and paper). It accounts for about 60 % of the total plant biomass produced on earth (Perez et al. 2002).

Lignocellulose is physically hard, dense and recalcitrant to degradation. However, it is an extremely rich and abundant source of carbon and chemical energy, therefore the recycling of carbon involving lignocelluloses is essential to maintain the global carbon cycle (Malherbe and Cloete 2002). Chemically, lignocellulose is a combination of two linear polymers, cellulose and hemicellulose and a nonlinear, three-dimensional polymer lignin (Perez et al. 2002). Cellulose is surrounded by matrix like hemicellulose and en-crusting lignin (Figure 3).

1.2.1. Lignin

Lignin is the most abundant high-molecular mass aromatic compounds in plants. High levels of lignin in plants are synthesized into wood and account for 15–36 % of the dry weight of wood whereas in grass it is less than 20 % . Lignin is complex of phenolic polymers that reinforce the walls of certain cells in the vascular tissues of higher plants


22

(Figure 4.) Lignin plays important roles in plants involving: mechanical support, wa-ter transport and in protecting cellulose and hemicelluloses from microbial attack by physical exclusion by reducing the sur-face area available of threse to enzymatic attack (Eriksson et al. 1990).

The lignin polymer arises from en-zyme-initiated oxidation of three phenolic precursors coumaryl, coniferyl and sinapyl alcohols, which differ in their degree of methoxylation. These precursors are syn-thesized from L-phenylalanine and L-tyro-sine, generated via the shikimic acid meta-bolic pathway, where the compounds are initially derived from CO2 fixed by plant photosynthesis (Higuchi et al. 1977). Lignin precursors and their relative amounts vary significantly between the plant species. Softwoods contain mainly guaiacyl lignin,

hardwood both guaiacyl and syringyl type lignin, whereas lignin in grasses consists of all three units (guaiacyl, syringyl and hydroxyphenol lignin) (Sjöström 1993).

Lignin formation results in an almost random series of bonding, and therefore the lignin polymer have no single repeating bond between these subunits.The most fre-quent inter-unit linkage is the β-O-4 (β-aryl ether). It is also the one most easily cleaved chemically, providing a basis for industrial processes such as chemical pulping (Singh 2006). The other linkages are β-5, β-β and 5-5 are more resistant to chemical degrada-tion (Sjöström 1993, Argyropoulos and Menachem 1997). At least 10 different types of aryl ether and carbon-carbon bonds are known to link phenylpropanoid units together (Argyropoulos and Menachem 1997). Recently a new type of linkage in softwood form-ing dibenzodioxocin moiety was discovered by Brunow and co-workers (Brunow et al. 2001).

Due to its complicated structure, lignin is highly resistant to microbial degradation and its association with cellulose and hemicellulose polysaccharides also imparts degra-dation resistance to these polymers (Hatakka 2001). Several properties of lignin account for its resistance to microbial attack: it is a water-insoluble, aromatic, three-dimensional molecule containing non-hydrolyzable bonds (Brunow 2001). Moreover, the enzymes needed for the complete degradation of lignin are only induced in the absence of readily available nutrients. Thus degradation of lignin is delayed and only occurs slowly.

The complex structure and the properties of the lignin polymers make studies on their degradation difficult. Isolation of native lignin is complicated if it is at all possible (Hatakka 2001) and therefore, suitable model compounds are needed for study. This problem can be overcome by using 14C-labeled lignin preparations, i.e. the dehydrogena-tion polymer (DHP). The chemical properties of DHP resemble those of natural lignin.

Figure 3. A schematic structure of lignocel-lulose


23

Figure 4. Schematic structure of spruce lignin, showing the common functional group (Bru-now 1998)

14C-labeled DHP can be prepared by polymerizing specifically or uniformly labeled con-iferyl alcohol with horseradish peroxidase (Kirk and Brunow 1988), resulting in guaiacyl (G-type) lignin. The G-type lignin - synthetic or natural is more recalcitrant to break-down than other types of lignin (Faix et al.1985). 14C-labeled synthetic lignins make it possible to follow and measure the fate of the 14C-label (mineralization, solubilisation) during microbial degradation.


24

Several chemical procedures have also been introduced for the estimation of lignin content (Tuomela et al. 2000). Some of these methods are more suitable for quantita-tive lignin analysis such as Klason lignin, Acid insoluble lignin, Kappa number, whereas others i.e., Kraft lignin more applicable for isolating lignin for biodegradation studies. The determination of Klason lignin is the most common method used to analyse lignin quantitively. In this method, hydrolysis of plant cell walls by sulphuric acid (70 % ) dis-solves all lignocellulose components other than lignin.The residual material is consid-ered to be lignin (Dence 1992). However, the Klason method is subject to errors if it is used for determining lignin content in plants that contain other interfering high mo-lecular weight substances such as proteins and tannins (Hammel 1997). The presence of humic substances (HS) formed during biological decomposition in compost or soil may also lead to error in the Klason lignin determination.

1.2.2. Lignin biodegradation

The biological degradation of lignin is an important contributor to the earth’s carbon cycle, because most renewable carbon is either in lignin form or in compounds protected by lignin from enzymatic degradation (Hatakka 2001). Lignin degradation is also respon-sible for wood destruction and may have an important role in plant pathogenesis (Lo-zoyova et al. 2006). On the other hand, lignin degrading organisms and their enzymes are of special interest and might be used in many industrial processes such as in pulp and paper technology and also for the treatment of many organopollutants, stains and dyes.

The most efficient lignin degrading microorganisms are taxonomically related to ba-sidiomycete white rot and litter decomposing fungi (Hatakka 2001). However, some ascomycetes, mitosporic, brown rotting and mycorrhizal fungi and some bacteria also contribute to lignin degradation (Daniel and Nilsson 1998, Hatakka 2001). Under aero-bic conditions, lignin is decomposed considerably but in anaerobic environments lignin losses are negligible (Kirk and Farrell 1987).

Unlike microbial degradation, abiotic degradation or transformation may also occur in special environments and under special conditions, such as those that arise from al-kaline chemical spills (Blanchette 1991) or UV radiation (Vähätalo et al. 1999). In a for-est ecosystem: temperature, moisture content and pH are the major factors influencing lignin transformation and breakdown activities (Donnelly et al. 1990 Criquet et al. 2000). The abiotic oxidation by transition metals such as Cu, Ni and Zn in calcareous soils also participates in the incorporation of phenolic and lignin related compounds into humus (Kaschl et al. 2002) and Mn-oxalate complex in cooperation with xylanase can modify the structure of plants cell wall (Lequart et al. 2000).

1.2.2.1. Lignin -degrading microorganisms

Although the carbon content in lignin is high, microorganisms are unable to utilize polymeric lignin as a sole source for carbon and energy (Kirk et al. 1976). It is generally believed that lignin depolymerization is necessary to gain access to cellulose and hemi-cellulose. Presumably, this is the real purpose for lignin biodegradation. During sugar utilization from polysaccharides of wood, H2O2 is produced by the action of glucose oxidase and glyoxyl oxidase (Kirk and Farrell 1987, Hatakka 2001) and this is a prereq-


25

uisite for degradation by white rot fungi. White rot fungi are the most efficient lignin degraders known so far. They can completely break down the lignin of wood by the enzyme-mediated oxidation of lignin referred as “enzymatic combustion” (Kirk and Farrell 1987). Fungal attack is an oxidative and non-specific process, which decreases methoxyl, phenolic and aliphatic content of lignin, cleaves aromatic rings and forms new carbonyl groups (Kirk and Farrell 1987, Hatakka 2001). These changes in the lignin mol-ecule result in depolymerization and carbon dioxide production (Kirk and Farrell 1987). The lignin degradation by white rot fungi is faster than that of other micro-organisms in nature. However it varies between species (Hatakka 2001).

White rot fungi secrete an array of extracellular enzymes i.e. lignin peroxidases (LiP), manganese peroxidases (MnP) and laccases. In many basidiomycetous white rot fungi, lignin degradation occurs during secondary metabolism, i.e. under conditions of nutri-ent limitation. The limiting nutrient for fungal growth in most wood and soils is prob-ably nitrogen (Kirk and Farrell 1987). It was suggested that N-limited growth conditions are natural for fungi, since wood contains only low levels of nitrogen (Kirk and Farrell 1987). However, there are variations in nitrogen metabolism between fungal species. The addition of organic nitrogen to the growth medium represses lignin-degrading activity in Phanerochaete chrysosporium (Keyser et al.1978) yet it stimulates biomass yields and laccase production in Bjerkandera sp. and Trametes pubescens (Kaal et al. 1993, Galhaup et al. 2002). Thus, lignin degradation is greatly influenced by the presence of nitrogen. Increasing the oxygen tension in cultures has a strong multiple activating effects on lignin degradation (Kirk and Farrell 1987). High oxygen levels (100 % ) enhance lignin mineralization in Phlebia radiata during growth on poplar wood (Hatakka and Uusi-Rauva 1983).

To date, little is known about the degradation of lignin by other microorganisms other than white rot fungi. Brown rot fungi which taxonomically belong to basidiomyc-etes minimally alter the lignin via hydroxylation and demethylation reactions that result in a loss of strength in the woody biomass along with a rapid loss of cellulose and hemi-cellulose (Blanchette 1995, Hatakka 2001). The presence of wood stimulated demethyla-tion activity of lignin model compounds by brown rot Gloeophyllum trabeum, which was able to evolved 30–60 % of 14CO2 from nonphenolic (4-O14CH3)-labeled β-O-4 dimer (Niemenmaa et al. 1992).

In a study using litter decomposing fungi from the genera Agrocybe and Stropharia the mineralization of 14C-(ring)-labeled synthetic lignin (DHP) was about half of the level obtained with white rot fungi (Steffen et al. 2000). Species such as Marismius quercophilus and Mycena inclinata were able to bring about a 60 % decrease in lignin content in oak leaves (Steffen et al. 2007) The most studied litter decomposing edible fungus Agaricus bisporus degrades as much as 35 % of lignin over an 80 day of cultivation period (Dur-rant et al.1991). Cyathus bulleri from the family Coprinaceae has been reported to degrade lignin (Abbott and Wicklow 1984) and because it produces low levels of cellulases and xylanases (Saxena et al. 1994) it is considered as a selective lignin degrader.

Some ectomycorrhizal fungi (Cenococcum, Amanita, Tricholoma and Rhizopogon) min-eralize 14C-labeled synthetic lignin and corn stalk lignin slowly. However, the efficiency of this process falls far behind that of white rot fungi (Trojanowski et al. 1984, Hasel-wandter et al. 1990).


26

Bacterial lignin degradation has been most extensively studied in filamentous actino-mycetes that belong to the genus Streptomyces. These gram-positive bacteria can solubilize less than 45 % of the total lignin present in water-soluble acid-precipitable polymeric lignin (APPL) and mineralize 3 % of the C-label over 21 days (Berrocal et al. 1997, Crawford et al. 1983). During growth in solid state culture of wheat straw Streptomyces cyaneus produces a laccase-type phenol oxidase and the activity of the enzyme was found to correlate with both solubilization and mineralization rates (Berrocal et al. 1997, Ber-rocal et al. 2000).

1.2.2.2. Lignin -degrading microfungi

The degradation of lignin by microfungi has only been studied by a few researchers. Some soil - and wood inhabiting fungi degrade lignin but the extent of degradation is limited compared to that of white rot fungi. The ascomycete Daldinia concentrica was able to bring about a 40 % decrease in lignin content (Nilsson et al. 1989) whereas Chrys-onilia sitophila decreased the lignin content of pine wood by only about 25 % (Ferraz and Duran 1995). Lignin is mostly modified by demethylation i.e. removal of methoxyl groups (Eslyn et al. 1975). Analysis of decayed wood after the fungal growth indicates that oxidative Cα –C β and β-O-aryl cleavages occurred during lignin degradation (Ferraz and Duran 1995).

Rodriguez et al. (1996b) observed a significant decrease in lignin in wheat straw by several ascomycetes and mitosporic fungi. Furthermore studies performed using isotop-ic methods confirmed the lignin degrading capacities of microfungi. Some degradation of differentially labeled DHPs by soft-rot microfungi of the strains Preussia, Chaetomium and Stachybotrys have been reported by Haider and Trojanowski (1975). The total release of 14CO2 in 10 or 15 days was only about 2–4 % of the total added radioactivity. Nev-ertheless, Chaetomium piluliferum released 30 % 14CO2 from differently labeled corn stalk lignin over seven weeks (Haider and Trojanowski 1980). In contrast to white rot fungi, microfungi preferably degrade DHP and corn stalk lignin in a high nitrogen medium (Haider and Trojanowski 1975, 1980). In similar degradation studies involving Penicil-lium chrysogenum, Fusarium oxysporum, F. solani and Pestalotia oxyanthi, the fungi were able to mineralize up to 2.5–9 % and 4.5–7 % of 14C-labeled lignin within 28 days, depending on the label in the synthetic lignin uniformly or side chain label (Rodriguez et al. 1994, Falcon et al. 1995 ). In contrast to white rot fungi, the degradation was maximal during primary metabolism. Some wood-rotting ascomycete species are also capable of miner-alizing lignin and lignin model compounds to some extent (Liers et al. 2006). This activ-ity has been reported for Xylaria species that are capable of causing white rot-like decay accompanied by substantial lignin loss (Pointing et al. 2003).

Several authors suggest that biodegradation of lignin by microfungi might be, at least partly, brought about by extracellular enzymes. In fact, different types of lignin-degrad-ing enzymes have been detected in several studies with ascomycetes. Thus, laccase was reported in Coniochaeta (Barbosa et al. 1996), Hortaea acidophila (Tetsch et al. 2005), Fusar-ium proliferatum (Regalado et al. 1999), Mauginiella sp. (Palonen et al. 2003), Penicillium chrysogenum (Rodriguez et al. 1996a) and Xylaria (Liers et al. 2006). Peroxidases have been reported for the ascomycete Chrysonilia sitophila (Rodriguez et al.1997), Aspergillus terreus LD- 1(Kanayama et al. 2002), Coniochaeta ligniaria NRRL 30616 (Lopez et al. 2007). How-


27

ever, these enzymes may not be so efficient in oxidizing lignin as those of white rot fun-gi, though they may have special properties. Hence, a thermophilic strain Thermoascus au-rantiacus, which degrades 15 % of lignin of Eucalyptus gradis and bleaches Eucalyptus kraft lignin (Machuca and Duran 1993, Machuca et al. 1998) produces high levels of phenol oxidase (Machuca et al. 1998). Phenol oxidase of T. aurantiacus has a capability to oxi-dize efficiently a range of substrates typical of phenoloxidases in the absence of H2O2 over an acidic pH range (2.6–3.0) and at elevated temperatures up to the 70–80°C range (Machuca et al. 1998). Fusarium proliferatum, which is able to mineralize synthetic lignin, also secretes superoxide radicals during lignin mineralization (Regalado et al. 1999). Su-peroxide radicals may generate highly reactive hydroxyl radicals, which are known to be involved in lignin degradation (Guillen et al. 2000). Only low levels of laccase activity were required for lignin degradation by Petriellidium fusoideum, where the activity was also correlated with the production of hydroxyl radicals (Gonzalez et al. 2002).

Fungi are also important in the degradation of lignin in aquatic habitats. Sutherland et al. (1982) reported 4–5 % mineralization of 14C-labeled maple and spruce lignin over 30 days by several species of marine fungi. The facultative marine ascomycete Sordaria fimicola mineralized 10 % of synthetic lignin and was able to produce lignin-modifying enzymes when grown in the low nitrogen medium supplemented with sea salts (Raghu-kumar et al. 1996).

1.2.3. Lignin degrading enzymes

1.2.3.1. Characteristic of lignin-degrading enzymes

Lignin biodegradation is a process involving the action of oxidative enzymes and in sub-sequent chemical reactions (Hatakka 1994, 2001). Reactions catalyzed by enzymes play a significant role in the complete degradation of lignocellulose biomass. Since the lignin polymer is large and highly branched, lignin-degrading mechanisms must be extracellu-lar and unspecific. The presence of stable ether and carbon-carbon bonds in lignin re-quires oxidative rather than hydrolytic enzymes. Due to the irregular structure of lignin the degradative enzymes must have lower substrate specificity than typical biological catalysts (Hammel 1997).

Some of the enzymes secreted by fungi generate hydrogen peroxide as an oxidant and others transfer the electrons. The most important lignin-modifying biocatalysts are lignin peroxidases (LiPs), manganese peroxidases (MnPs), functional hybrids of both enzymes (versatile peroxidases VP) and laccases (phenol oxidases). All extracellular per-oxidases and laccases have the ability to catalyze one-electron oxidation resulting in the formation of radicals, which undergo several spontaneous reactions. These, in turn lead to various bond cleavages including aromatic ring fission (Kirk and Farrell 1987, Hatak-ka 2001). Apparently, these enzymes act using low-molecular mass mediators to carry out lignin degradation.

1.3.2.2. Peroxidases

LiP and MnP are heme-containing proteins, which require hydrogen peroxide as an oxi-dant. The lignin-degrading system depends on low molecular mass metabolites and co-factors. A secondary metabolite, veratryl alcohol (3, 4-dimethoxylobenzene) is a redox


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mediator for LiP, whereas Mn+2, which is ubiquitous in all lignocelluloses and in soil is a redox mediator for MnP. Some of most important features distinguishing these enzymes from other oxidoreductases are their very low pH optima and much higher redox poten-tials (Hatakka 2001, Hofrichter 2002).

LiP was first found in the lignin-degrading fungus Phanerochaete chrysosporium dur-ing secondary metabolism under nutrient-limited culture conditions. LiP is produced by several white rot fungi such Phlebia radiata (Hilden et al. 2006), Trametes trogii (Vares and Hatakka 1997) and Bjerkandera sp. BOS55 (ten Have et al. 1998). To date, there is limited knowledge on LiP production in fungi other than white rot basidiomycetes. However, three isoforms of LiP has been purified from the ascomycete Chrysonilia sitophila (Rod-riguez et al. 1997). LiP-like peroxidase from the lignite degrading fungus Penicillium decum-bens P6 has recently been characterized (yang et al. 2006).

The substrates of LiP include both phenolic and non-phenolic aromatic compounds. The phenolic substrates are oxidized to yield products similar to those produced by per-oxidases, while oxidation of nonphenolic methoxybenzenes is unique to LiP (Kersten et al. 1985). The oxidation of these substrates to yield aryl cation radicals can result in either demethylation, Cα- Cβ cleavage of lignin model compounds, benzylic alcohol oxi-dation, or hydroxylation of aromatic rings and side chains (Kirk and Farrell 1987).

MnP is secreted by a distinct group of wood white rot and soil litter basidiomycetes. However, recently Kanayama et al. (2002) purified an alkaline MnP-like peroxidase from the ascomycete Aspergillus terreus LD-1. This enzyme seems to be very attractive to the pulping industry since it has unique pH optima of between pH 11 and 12.5.

MnP oxidizes Mn2+ to Mn3+ using H2O2 as the oxidant. The product of Mn+2 oxida-tion, Mn+3 must be chelated by organic acids such as oxalate or malonate , which are pro-duced by the fungus (Galkin et al. 1998, Hofrichter et al. 1999). With the help of these chelators Mn+3 ions are stabilized promoting their release from the enzyme into materi-als such wood. Chelated Mn +3 acts as a strong oxidant that preferably attacks phenolic moieties of lignin resulting in the formation of free radicals that tend to disintegrate spontaneously (reviewed by Hofrichter 2002). However, in the presence of different un-saturated fatty acids and their derivates, nonphenolic lignins are oxidized through a MnP - lipid system (Kapich et al. (1999a, 1999b). Unlike lignin, purified MnP also oxidizes HS from litter and brown coal (Steffen et al. 2002, Hofrichter and Fritsche 1997) and HS synthesized from catechol (Hofrichter et al.1998, Steffen et al. 2002) in addition to sev-eral organopollutants (Steffen et al. 2003).

The third type of peroxidase called versatile peroxidase (VP) has also been reported to be secreted from Pleurotus eryngii (Camarero et al. 1999) This peroxidase is capable of the oxidative reaction, characteristic of both LiP and MnP.

1.3.2.3. Laccase

Laccase is a phenol oxidase, which belongs to the blue multicopper oxidases. These en-zymes catalyze one-electron oxidation of four reducing-substrate molecules concom-itantly with four-electron reduction of molecular oxygen to water. Laccases typically contain four copper atoms of three types that can be identified on the basis of their spectroscopic and paramagnetic properties. The presence of different copper domains are important for the catalytic activity of laccases. The type-1 Cu bound via two His and


29

one Cys as ligands, functions as the primary electron acceptor. This extracts electrons from the reducing phenolic substrates and transfers them to the trinuclear centre at the Type-2 and Type-3 Cu sites. The trinuclear centre is typically coordinated by eight His residues and is the binding site for the second substrate, i.e. molecular oxygen. This oxy-gen atom accepts electrons from the Type-1 Cu site for its subsequent reduction to water (Claus 2003, Baldrian 2006; Figure 5).

A typical laccase has a molecular mass of about 60–80 kDa. However, enzymes from ascomycetes Monocillium indicum and Gaeumannomyces graminis appear to be substantially larger with molecular mass of 100–190 kDa (Thakker et al. 1992, Edens et al. 1999). Laccase mostly exhibits isoelectric points (pI) and pH optima in the acidic pH range (Bollag and Leonowicz 1994). However, laccases of some soil-inhabiting basidiomycetes (Schneider et al. 1999) and ascomycetes (Chefetz et al. 1998a, Robles et al. 2000) includ-ing Rhizoctonia praticola (Bollag and Leonowicz 1994), Coprinus cinereus (Schneider et al. 1999) and Chaetomium thermophilum (Chefetz et al. 1998a) have higher pH optima over a 7–8 pH range. Temperature profiles of laccase shows optima ranging between 30–60°C (Gianfreda et al. 1999).

Laccases are mostly inducible enzymes and their induction has been observed at the level of transcription and translation upon addition of copper, and also aromatic com-pounds such xylidine (Collins and Dobson 1997, Palmieri et al. 2000, Litvintseva et al. 2002, Tetsch et al. 2005). However, the repression of laccase at the high concentrations of these compounds due to their toxic effect has also been demonstrated in some fungi (Bollag and Leonowicz 1984, Eggert et al. 1996).

Natural and synthetic lignin in addition to industrial lignins such as lignosulfonates or indulin AT are good elicitors of laccase production. Lignocellulosic residues in growth media significantly increase laccase formation in several fungi (Ardon et al.1996, Machu-ca et al. 1998, Pickard et al. 1999, Lorenzo et al. 2002).

Laccase has a rather low specificity as regards to reducing substrates. Therefore a number of quite different organic and inorganic compounds including diphenols, polyphenols, substituted phenols, diamines and aromatic amines are readily oxidized (Thurston 1994). Laccase catalyzes the cleavage of the Cα- Cβ bonds in phenolic β-1 and β-O-4 lignin model dimers by oxidizing the Cα and by splitting the aryl-alkyl bonds (Eriksson et al. 1990).

Laccase in not a key enzyme in lignin degradation, as lignin contains only 10–15 % of phenolic structures in wood (Singh 2006). However, in the presence of aromatic elec-tron-transfer mediators such as ABTS, the blue laccase from Coriolus (Trametes) versicolor becomes capable of oxidizing non-phenolic substrates (Bourbonnais and Paice 1990). The same effect was exerted by the laccase secreted by Pycnoporus cinnabarinus in the pres-ence of the fungal metabolite 3-hydroxyanthranilate (Eggert et al. 1996).

Laccases are produced by higher plants and fungi, but they are also found in bacteria, yeasts and insects (Thurston et al. 1994, Claus 2003). In plants, laccases are involved in the formation of lignin, whereas in bacteria laccases are involved in melanin production and spore coat resistance (Castro-Sawinski et al. 2002, Martins et al. 2003).

Laccase-like activities have also been detected in the cuticle of larval and adult stage of insects, during the sclerotization process (Hopkins and Kramer 1992).


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Figure 5. Catalytic cycle of laccase modified from Baldrian (2006)

The majority of laccases characterized so far have been derived from fungi especially from white rot basidiomycetes, ascomycetes and mitosporic fungi. Well-known laccase producers are phytopathogenic ascomycetes such as Botrytis cinerea (Slomczynski et al. 1995), Gaeumannomyces graminis (Edens et al. 1999), Magnaporthe grisea (Iyer and Chattoo 2003), Ophiostoma novo-ulmi (Binz and Canevascini 1997) and Mauginella sp. (Palonen et al. 2003). Laccase production was also reported for some: soil ascomycete species from the genera Aspergillus, Fusarium and Penicillium (Rodriguez et al. 1996b, Scherer and Fischer 1998, Regalado et al. 1999), compost Chaetomium thermophilum (Chefetz et al. 1998a ), freshwater ascomycetes (Abdel-Raheem 1997, Junghanns et al. 2005) and from lichenized ascomycetes of the order Peltigerales (Laufer et al. 2006, Zavarzina and Zavar-zin 2006). The crystal structure of laccase found in ascomycete Melanocarpus albomyces has already been solved (Hakulinen et al. 2002).

The wood-degrading ascomycete Bothryosphaeria that is closely related to the wood-rotting basidiomycetes constitutively produces a dimethoxyphenol-oxidizing laccase (Vasconcelos et al. 2000 ), which is significantly induced by veratryl alcohol (Barbo-sa et al. 1996, Dekker et al. 2001). Several ascomycete species involved in the decay of plant biomass in terrestrial habitat and salt marshes have been shown to have lac-case genes and to oxidize syringaldazine (Lyons et al. 2003, Pointing et al. 2005). Trichoderma strains have also been reported to produce syringaldazine-oxidizing polyphe-noloxidases (Assavanig et al. 1992). These are mainly associated with spores, which may act in the morphogenesis of this fungus (Assavanig et al., 1992; Hölker et al., 2002). Two Xylaria strains i.e., X. polymorpha and X. hypoxylon exhibit ABTS oxidizing laccase in com-plex liquid media and solid birch wood cultures (Liers et al. 2006).

Fungal laccases also contribute to pigment production, fruiting body formation and plant pathogenesis (Thurston 1994, Gianfreda et al.1999). They are found to be involve in various ecological processes in soil, forest litter and compost environments and they have often been isolated from these habitats (Criquet et al. 2000, di Nardo et al. 2004, Chefetz et al. 1998b). In the soil and compost environments, released laccases are capa-ble of both polymerization and depolymerization of humic acids, and may contribute to carbon cycling (Stevenson 1994, Chefetz et al. 1998b, Zavarzina et al. 2004). Soil orga-


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nopollutants may also be oxidized by laccase to less toxic polymers, which after various transformation may be incorporated in the soil humus (Gianfreda and Bollag 1994).

1.2.4. Cellulose and hemicellulose

Cellulose and hemicelluloses act as both structural and energy-storage components of plants. Cellulose is the most abundant, insoluble and highly ordered renewable poly-mer found in nature (Lynd et al. 2002). It is the major constituent of plant cell walls providing their rigidity (Beguin and Aubert 1994). Cellulose comprises approximately 30–40 % of dry wood weight and 45 % of the dry weight of grasses. The polymer consists of D-glucose subunits linked by β-1, 4 bonds, forming long insoluble chains (microfibrils) linked together by hydrogen bonds and van der Waals forces. The micro-fibrils are grouped together to make up the cellulose fiber. The degree of polymeriza-tion of cellulose chains ranges from 500 to 25 000 (Kuhad et al. 1997). Cellulose is often crystalline in the native stage and is surrounded by a mixture of amorphous cellulose (non-organized chains), hemicellulose and lignin. Because of its structural rigidity, crys-talline cellulose is resistant to the action of individual cellulases. Effective conversion of cellulose to monosaccharides is therefore only possible by the synergistic action of these enzymes (Figure 6). Amorphous regions occur near the crystal surface and are prone to enzymatic attack (Beguin and Aubert 1994). The crystallinity index, and the nature of substances (lignin) with which the cellulose is associated are the most important factors affecting the speed of cellulose degradation (Kuhad et al. 1997).

Hemicelluloses, the third most abundant constituents of plant cell walls found in na-ture, represent about 20 to 35% of the lignocellulose dry mass. They are heterogeneous polymers of pentoses (D-xylose, L-arabinose), hexoses (D-mannose, D-glucose, and D-galactose) and sugar acids. They are linked together by β-1,4- glycosidic bonds, but β-1,3-, β-1,6-, α -1,2-, α- 1,3- and α -1,6- glycosidic bonds are also reported (Sjöström 1993). Hemicelluloses are chemically associated with or cross-linked to other polysac-charides, proteins and lignin.Moreover, they form a matrix together with pectin and pro-teins in primary cell walls and with lignin in secondary cell walls. The matrix of lignin and hemicellulose encrusts and protects the cellulose of the plant cell wall (Hammel 1997).

The major hemicelluloses of hardwood and annual plants are xylans (15–30% ), which probably interact with lignin and cellulose more than any other hemicellulose (Kuhad et al. 1997). The main hemicelluloses of softwood are galactoglucomannans (15–20% ), arabinoglucoroxylan, and arabinogalactan (Sjöström 1993). Since hemicelluloses have an amorphous nature and a lower degree of polymerization (approximately 70–200) they are degraded more easily than cellulose (Kuhad et al. 1997, Perez et al. 2002).

1.2.5. Cellulose and hemicellulose biodegradation

The ability to produce cellulolytic enzymes is widespread among microorganisms, but only a limited number of species are actually able to degrade native cellulose in its crys-talline form. Cellulolytic microorganisms (fungi and bacteria) can establish synergistic relationships with non-cellulolytic species in cellulosic wastes (Maheshwari et al. 2000). The interaction between both populations leads to a complete degradation of cellulose,


32

which releases CO2 and water under aerobic conditions, or carbon dioxide, methane and water under anaerobic conditions (Béguin and Aubert 1994).

The cellulolytic enzyme system of fungi consists of a number of extracellular endog-lucanases, cellobiohydrolases and β-glucosidases, which work together catalyzing the hy-drolysis of cellulose (Lynd et al. 2002). Endoglucanases (EGs) represent less than 20 % of the total protein in Hypocrea jecorina (anomorph Trichoderma reesei) and preferentially hydrolyse cellulose microfibrils in the amorphic parts of the fibril, releasing new termi-nal ends. Cellobiohydrolases (CBHs), which may account for 20 to 60 % of the total cellulase proteins of fungi act on the existing or endoglucanase-generated chain ends. In H. jecorina CBH I attacks reducing ends and CBH II the non-reducing ends of the fiber (Teeri 1997). CBHs and EGs can degrade amorphous cellulose, but CBHs are the only enzymes that efficiently degrade the crystalline form of cellulose. Both enzymes release cellobiose molecules (reviewed by Teeri 1997). The effective hydrolysis of cellulose also requires β-glucosidases (BGL), which break down cellobiose and release two glucose molecules. Products of cellulose degradation are made available as carbon and nitrogen sources for either cellulolytic organisms, or other microbes living in the environment containing cellulosic materials.

Fungal cellulases are the most widely investigated so far. Cellulases from microfungi have been studied more than those of any other physiological group, and their cellulases

currently dominate the industrial applications of cellulases (Nieves et al. 1998). In par-ticular, the cellulase system of Hypocrea jecorina, (formerly known as Trichoderma reesei)) has been the focus of research for 50 years. The full genera sequence of this fungus is pub-lished. H. jecorina produces at least two cellobiohydrolases (CBHI and CBHII), five en-doglucanases (EGI, EGII, EGIII, EGIV, and EGV), and two β-glucosidases (BGLI and BGLII; Kubicek and Penttilä 1998, Nevalainen and Penttilä 2004). The cellulase system of the thermophilic fungus Humicola insolens is homologous to the H. jecorina system and also contains several enzymes (Schülein 1997). Figure 6. A simplified schematic of complete enzymatic hydrolysis of a cellulose microfi-bril by fungi modified according to Lynd et al. (2002)


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A 28-kDa endoglucanase (EG28) from white rot fungus Phanerochaete chrysosporium has different properties from the common type of cellulase (Henriksson et al. 1999). EG28 lacks the ability to bind crystalline cellulose and is active with xylan and mannans, suggesting a possible role in lignocellulose degradation. The P. chrysosporium genome re-veals an impressive array of genes encoding cellulases including 40 endoglucanases, sev-en cellobiohydrolases and at least nine β-glucosidases (Martinez et al. 2004).

Cellulases are inducible enzymes, and their biosynthesis is enhanced markedly in me-dia containing inducers (Béguin and Aubert 1994, Aro et al. 2005). Low constitutive amounts of cellulases can be found in saprophytic fungi, which makes it possible for them to attack cellulose when available and thus lead to the formation of the inducer activity of cellulase biosynthesis. Therefore the transcription of H. jecorina EG and CBH genes present in the low levels prevailing under in the non-induced conditions can be induced up to at least 1100-fold in the presence of cellulose (Carle-Urioste et al. 1997). Cellobiose and sophorose (β-1, 2-glucobiose) are also good inducers, whereas glucose is a strong repressor of endoglucanases activity (Béguin and Aubert 1994, Ilmén et al. 1997, Kubicek and Penttilä 1998). When rice and wheat straw were used as carbon sources Neurospora crassa, Melanocarpus sp. MTCC 3922, Scytalidium thermophilum MTCC 4520 and Myceliophthora sp. IMI 387099 were able to initiate cellulase production(Romero et al. 1999, Kaur et al. 2006, Badhan et al. 2007). In contrast, when ammonium salts were used as nitrogen source cellulase production in Aspergillus fumigatus and Thermoascus auran-tiacus were initiated (Steward and Perry 1981, Kalogeris et al. 2003).

In fungi the production of cellulases may also be regulated by factors other than in-duction and repression by sugars. Several lignin-related aromatic compounds, which are found in association with cellulose in nature, stimulate or inhibit cellulase production. This feature is common to some wood-rotting white and brown rot basidiomycetes such as Phlebia radiata, Trametes gibbosa, Trametes (Coriolus) versicolor, Postia placena and Gloeophyllum trabeum (Müller et al. 1988, Highley and Micales 1989, Tsujiyama et al. 2003).

The hydrolysis of hemicellulose requires similar types of enzymes to those required for cellulose hydrolysis. However, more enzymes are required for the complete degra-dation of hemicellulose, because of the greater complexity of hemicellulose compared to cellulose (Malherbe and Cloete 2002). Hemicellulases hydrolyze glycosidic linkages in hemicelluloses and are classified according to their substrate specificities. The total enzymatic degradation of hemicellulose polymers requires the action of “endo-type” enzymes that liberate short oligosaccharides, which are subsequently degraded by side-group cleaving enzymes and “exo-type” enzymes and finally monomeric sugars and acetic acid are formed. Similar to the cellulases, hemicellulases act synergistically. Of all these enzymes xylanase is the best studied (reviewed by Kuhad et al. 1997).

Xylanases have been found in many ecological niches, where plant material is present. Complete breakdown of a branched xylan requires the action of several enzymes, partic-ularly endo-1,4-β-xylanases and β-xylosidases. Endoxylanases are able to cleave the xylan backbone into smaller oligosaccharides including xylobiose, which can then be degraded further by β-xylosidases into D-xylose (Biely and Tenkanen 1998). Both classes of en-zymes ain addition to their encoding genes have been found in many microorganisms. Xylanases of Trichoderma spp. and Aspergillus spp. are the most studied (reviewed by Bie-ly and Tenkanen 1998 and by de Vries and Visser 2001). However, xylanases have also


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been described in several basidiomycetous white rot (Rogalski et al. 1993, Maijala 2000, Khalil 2002), brown rot (Ritschkoff et al. 1994) and in litter decomposing fungi (Steffen et al. 2007). As in the mesophilic fungi, a multiplicity of xylanases have been observed in some thermophilic fungi (Prabu and Maheswari 1999, Puhart et al. 1999, Latif et al. 2006, yang et al. 2006, Badhan et al. 2007). Xylanases are the major group of industrial enzymes, that finds significant application in the paper and pulp industry. This is because the hydrolysis of xylan facilitates the release of lignin from paper pulp and hence reduc-es the usage of chlorine as the bleaching agents (Buchert et al. 1998).

1.3. Humic substances

1.3.1. Occurrence and formation of humic substance

The term humic substance(s) (HS)refers to high-molecular mass, dark brown material that is rich in aromatic compounds. It originates from plant, animal or microbial organic matter. They are the most widely distributed biosynthesizes products on the earth. Be-sides soils, humic substances (HS) can be found in lakes, rivers, compost, sediments, peat and lower-rank coals (Aiken et al. 1985).

HS comprise three fractions that are humic acids (HAs; Figure 7), fulvic acids (FAs; Figure. 8) and humin. The fractions differ in acid and base solubility. The physico-chem-ical characteristics of HS are shown in Table 5. HS mainly consist ofmolecules with aro-matic rings connected by long aliphatic chains (Shevchenko and Bailey 1996). Moreover, HS fractions are presumed to have similar molecular structures, only showing differ-ences in the degree of cross-linking between their macromolecules, the content of oxy-gen and the amount of hydroxyl, carbonyl or carboxyl groups (Kästner and Hofrichter 2001).

Lignin and polysaccharides are two major organic precursors of HS formation (In-bar et al. 1989). The glycosidic link between lignin and polysaccharides is broken first. Changes in lignin begin with the loss of phenolic and methoxyl groups with an increase in carboxyl and carbonyl groups (Inbar et al. 1992, Shevchenko and Bailey 1996). Thus HS are less aromatic and contain fewer methoxyl but more carboxyl groups than lignin (Shevchenko and Bailey 1996). The process in which organic matter is transformed into humic substances (HS) is called humification. In composts the process forms humus-like materials which are permanent. Humification starts as early as the first mesophilic phase of composting, develops in the thermophilic phase and finally results in the pro-duction of humic substances in the maturing phase of the composting process. How-ever, mature compost is only an intermediate product of humification because HS still undergo changes. Decomposition of high and low-molecular-weight plant components and synthesis of microbial cell constituents are involved in humification. During com-posting HS are formed in a relatively short period of time.

The humification of lignocellulosic waste depends on lignin oxidation, which mainly occurs during the thermophilic phase of composting (Baca et al. 1992). Phenoloxidases (peroxidases and laccase) catalyze this process by mediating oxidative coupling of phe-nolic products that result from biomass decomposition (Chefetz et al 1998a, Dec and Bollag 2000, Zavarzina et al. 2004). During plant degradation 70 % of organic carbon is mineralized. At the same time lignin is covalently bound to HS (Shevchenko and Bai-


35

ley 1996, Almendros et al. 2000). Humification reactions still continue, after molecules chemically bound together. Thus, lignin is in fact being degraded at the same time as some of it is being polymerized (Shevchenko and Bailey 1996). In the beginning of the composting process HAs are formed from aliphatic compounds, which are later re-placed by aromatic molecules with a high proportion of oxygen and nitrogen constitu-ents (Miikki et al. 1997, Veeken et al. 2000, Sanchez-Monedero et al. 2002). Compost HAs are less aromatic, contain fewer carboxyl and oxygen-containing functional groups and they have lower molecular mass than soil HAs (Inbar et al. 1990, 1992, Garcia-Gil et al. 2004, Liguirati et al. 2005). Elemental composition, functional group contents and E4 / E6 ratio (indicate changes in molecular mass) analyses of HAs from various composts and soil are shown in Table 6. The composition of compost HAs resembles those of plant residues, peat and incompletely humified materials (Inbar et al. 1990, 1992).

Humic acids represent the most abundant fraction of humic substances. HAs are a complex of polymers in which phenolic and aliphatic units are linked by peptides, amino acids, amino sugars and other organic constituents. HAs can be recovered from soil, litter, compost or low-rank coal such as lignite or brown coal; as sodium salts (Na humates) by sodium alkaline (Hofrichter and Fakoussa 2001a). These are subsequently precipitated with hydrochloric acid at pH 2 (Senesi and Loffredo 2001). Due to HAs high molecular mass and heterogeneous structure comprising aromatic building blocks, they are resistant to microbial attack (Haider and Martin 1988, Willmann and Fakoussa 1997b).

1.3.2. Biodegradation of humic substances

Resistance to biodegradation is one of the most important properties of humic sub-stances. Since soil humus generally decomposes at the rate of 2–5 % a year (Linharse and Martin 1978), the age of the HS seems to play an important role for the metabo-lism and mineralization of these compounds. Because of their size, humic compounds cannot be taken up into the hyphae, thus an extracellular enzymatic activity is assumed (Kästner and Hofrichter 2001) These enzymes may either polymerize HS under certain conditions in addition to degrading them (Chefetz et al. 1998a, Zavarzina et al. 2005).

A variety of techniques can be used to follow the bioconversion of HS such as microbiological, spectrophotmetric, gravimetric and radioactive methods. Nowadays, one of the best and easiest methods available to study the degradation of HS is the ap-plication of synthetic radioactively labeled HS preparations. Synthetic HS are prepared from 14C-labeled polyphenols such as 14C-labeled 14C-catechol, 14C-glucose or 14C-glycine by the oxidative polymerization or the Millard reaction (Blondeau 1989, Hofrichter et al. 1998b). However, it should be kept in mind that the synthetic model of HS may differ from the naturally occuring HS. Moreover, the mineralization of synthetic compounds does not reflect the whole degradation process, which occurs in natural environments. Extracted natural HS can also be used in degradation studies and these are often ana-lysed by HPSEC (high pressure size exclusion chromatography). HPSEC is used to eval-uate changes in the relative molecular weight distribution of soluble HS and gives indica-tions of the absolute molecular masses of humic and fulvic acids (Blondeau 1989).


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Numerous organisms are able to slowly decompose humic acids slowly. The most active degraders are basidiomycetous fungi and actinobacteria, which are also efficient lignin degraders.

Degradation of HA is similar to that of lignin, that occurs under co-metabolic con-ditions, i.e. in the presence of easily assimilable and metabolizable carbon sources such as glucose and pentoses (Blondeau 1989). However, exceptions exist (Řezàčovà et al. 2006).

The release of 14CO2 from 14C-HA and decolourization i.e. conversion of high mo-lecular-mass (MM) HA into low molecular-mass FA of natural HA under co-metabolic conditions was observed in liquid glucose-mineral salt cultures of the basidiomycetes Phanerochaete chrysosporium and Trametes versicolor (Haider and Martin 1988, Blondeau 1989, Dehorter and Blondeau 1992), Collybia dryophila (Steffen et al. 2002) and the actinobacte-rium Streptomyces viridisporus (Kontchou and Blondau 1992). These were found to degrade HAs and form lower molecular mass FAs. The first successful study on the degradation of HAs was performed by Hurst et al. (1962), who reported that wood-colonizing basid-iomycetes such as T. versicolor and Hypholoma fasciculare were capable of decolourizing soil HA. Soil HA is also decolourized in the presence of an additional carbon source such as glucose by isolates of Streptomyces (Kontchou and Blondeau 1992, Dari et al. 1995). In bacterial cultures the decolourization was partially caused by adsorption of HA onto the

Humic substance (HS)

Color Solubility Molecular mass (MM) kDa

Humic acid (HA) dark brown soluble in alkali 1.4–100Fulvic acid (FA) yellowish soluble in alkali

and in acid0.5–30

Humin black insoluble in alkaliand in acid in all pH

similar to MM of HA

Figure 7. (above) Model structure of hu-mic acid Stevenson (1994)

Figure 8. (right) Structural models of se-lected subfractions of fulvic acids according to Leenheer & Rostad (2004; MM = molec-ular weight in Daltons).

Table 5. Chemical and physical characteristics of humic substances (Stevenson 1994, Shevchenko and Bailey 1996, Kästner 2000)


37

bacterial cells surface, and partially caused by extracellular non-selective enzymes (Adhi et al. 1989).

The efficiency of fungi in modifying humic substances is considered to be associ-ated with their extracellular non-specific enzyme system, especially lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase (Kästner and Hofrichter 2001). Addi-tion of HA to fungal growth media may either stimulate enzyme activities (Dehorter and Blondeau 1992, Willmann and Fakoussa 1997b, Steffen et al. 2002) and RNA ex-pression (Scheel et al. 2000), or inhibit them (Ralph and Catcheside 1994). Thus, in soil the absorption of laccase on humic constituents may change enzyme availability and ac-tivity (Criquet et al. 2000, Claus and Filip 1990, Kang et al. 2002). However, Keum and Li (2004) suggest that the inactivation of laccase by HA is not due to binding to humic material but to the dissociation and chelation by HA of copper from the active centre of the enzyme.

Table 6. Elemental composition, functional group contents and E4 / E6 analysis of humic acids (HAs) from different composts and soil (Aiken et al. 1985, Chen et al. 1996, Unsal and Ok 2001)

Constituent Humic acids origin

CompostsElemental com-position

Municipal solid waste (MSW)

Sewage sludge (SS)

Wood compost (WC)

Grape waste (GW)

Average of different soils

C ( % ) 52.0 53.0 50.6 58.2 56.2

H ( % ) 6.0 5 5.6 5.9 4.7

O ( % ) 37.2 36.7 nd 29.0 32.2

N ( % ) 6.3 4.2 3.5 5.8 1.9

H / C a 1.4 1.2 1.3 1.2 1

C / N 11.7 13 16.9 14.2 20

Total acidityb 13.5 21.9 16.3 13.1 6.7

COOHd

(meq g-1)1.9 1.2 2.2 1.7 3.6

Phenolic-OHd

(meq g-1)11.6 20.7 14.1 11.4 39.0

E4 / E6* 7.1 2.2 4.5 3.3 2.1

a represents HA aromaticity (a low ratio indicates an aromatic structure)b sum of carboxyl and phenolic groups* E4 / E6 (the ratio between absorbance at 465 nm and 665 nm, the higher ratio indicates a lower molecular mass of HA)d functional groupnd = no data


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High activities of ABTS and syringaldazine oxidizing enzymes (MnP, laccase) de-tected during the process of HA depolymerization by Nematoloma frowardii b19 in the agar medium, indicates the direct involvement of lignin-modifying enzymes in the deg-radation process (Hofrichter and Fritsche 1997). However, it is interesting to note that depolymerization occurred over the course of several days. Other authors have demon-strated the important role of white-rot laccases in the disintegration of HA. (Frost and Fakoussa 1999, Zavarzina et al. 2004).

Among all lignin-modifying biocatalysts involved in HA degradation, MnP is consid-ered to be a key enzyme. The ability of MnP derived from white rot fungus Nematoloma frowardii to mineralize humic macromolecules in vitro has been demonstrated in study us-ing 14C-HA prepared from 14U-catechol (Hofrichter et al. 1998b). Approximately 17 % of 14C-labeled HA is released as 14CO2 within one week of enzymatic treatment. Other in vitro studies (Wunderwald et al. 2000, Steffen et al. 2002) confirm that MnP is able to de-polymerize and mineralize HAs. A positive correlation between decolourization of HA and MnP production has been also reported in many wood and straw degrading basidi-omycetes (Gramss et al. 1999). However, the brown rot fungus Fomitopsis pinicola, which does not produce extracellular lignin-degrading enzymes can alter molecular masses of HA as effectively as the wood-decaying white rot fungus Hypholoma (Nematoloma) frowar-dii by producing lignin-modifying enzymes (Gramss et al 1999).

The role of white rot fungi in the degradation of humus in nature is unclear. White rot fungi grow preferentially in compact wood but they compete poorly with soil and compost-dwelling microorganisms. Moreover, soil HS were even found to inhibit the growth of white rot fungi (Rayner and Boddy 1988). The pH in soils and compost is usually higher and comparable to that in wood whereas the C/N ratio is lower than re-ported for optimal degradation of lignin and HAs by wood-colonizing white rot fungi. However, two white rot fungi isolated from biosolids compost namely Trametes sp. M23 and Phanerochaete sp.y6, have the ability to bleach compost HA during growth under solid- state conditions using perlite as a solid support (Granit et al. 2007). The strong bleaching ability of coal derived HA (lignite) by these fungi has been reported previously by Frost and Fakoussa (1999).

Litter decomposing fungi, which are taxonomically and physiologically related to white rotting fungi that occupy soil and humus layers of forests have also been shown to disintegrate high-molecular mass HS of forest litter efficiently (Steffen et al. 2002). Colly-bia dryophila can degrade both soil HA and synthetic 14C-HA prepared from catechol and it secretes MnP that is involved in the mineralization process. In contrast, only traces of a synthetic HA-like polymer were mineralized by the ectomycorrhizal symbionts of Douglas fir (Pseudotsuga menziesii [Mirb.] Franco; Durral et al.1994). Nevertheless, humic substances stimulate root colonization and the production of mycelium in the mycor-rhizal fungus Glomus claroideum BEG 23 (Gryndler et al. 2005).

The utilization of HA has so far been studied in wood and litter-decomposing ba-sidiomycetes (Blondeau 1989, Dehorter and Blondeau 1992, Steffen et al. 2002) whereas the involvement of microfungi in this process has been studied only rarely. According to Kästner and Hofrichter (2001) none of the six Penicillium sp. strains isolated from differ-ent soils was capable of utilizing HA as the sole carbon source. In another study Aspergil-lus awamori, Penicillium sp. and Humicola (Thermomyces) insolense could decolourize forest soil


39

HA under co-metabolic conditions and HA was also utilized as the sole carbon and /or nitrogen source by the fungi (Mishra and Srivastava 1986). Řezàčovà et al.(2006) did not find a significant effect of additional carbon source on the utilization of soil HA by microfungi. Gramss et al. (1999) detected the decrease of decolourization rates by about 25–50 % in media containing HA as the sole carbon source compared to supplemented media with malt extract. Higher fungal biomass and decolourization of litter HA by Cha-lara longipes has been observed in media supplemented with nitrogen (Koukol et al. 2004). Aquatic HA was degraded under co-metabolic conditions by Cladosporium cladosporioides isolated from bog lake water (Claus and Filip 1998). The degradation process was ac-companied by an increase in the oxygen content of HS as well as by a relative decrease in aromatic carbon and an increase in aliphatic carbon (Claus and Filip 1998).

Laccase from C. cladosporioides does not decolourize HA to a significant extent only (5 to 10 % ) even in the presence of enzyme mediator HBT (Claus and Filip 1998). The as-comycetes Xylaria sp i70, Botrytis cinerea H1 and mitosporic fungus Alternaria sp.G5 also produced extracellular enzymes, although they are not able to effect coal HA decolouri-zation (Hofrichter and Fritsche 1996). However, Paecilomyces farinosus produced laccase and decolourized coal HA as efficiently as did some white rot fungi such as Pleurotus os-treatus (Frost and Fakoussa 1999). The ability to mediate the extensive solubilization of select low rank coals has also been reported for non- lignin degrading fungi such as As-pergillus spp., Paecilomyces sp., Penicillium waksmani and Candida spp. (Ward 1985, Scott et al. 1986, Steward et al. 1990). Recently, yang et al. (2004) purified LiP-like peroxidase from the lignite-degrading microfungus Penicillium decumbens, which most probably is involved in the biodegradation process. The decolourizing/depolymerizing effect of purified LiP from Phanerochaete chrysosporium on coal macromolecules has been demonstrated earlier by Ralph and Catcheside (1999). Extracellular peroxidase, esterase and phenoloxidase enzymes also appear to be involved in coal solubilization by Trichoderma sp. and Penicil-lium sp. (Laborda et al. 1999). Interestingly, none of these enzymes were detected when fungi were grown in the absence of coal, which may indicate that they are coal-induced enzymes.

2. AIMs OF THe sTUDy

The main aims of the work were:(i) to examine of the ability of the compost-dwelling fungus Paecilomyces inflatus to modify and degrade the lignocellulose complex by each single components in addition to the humic substances under different growth conditions,(ii) to identify and partially characterize the main extracellular lignocellulose degrading enzymes secreted by the fungus during biodegradation process, (iii) to examine the degradation strategies of different P. inflatus strains under various conditions.


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3. MATeRIALs AND MeTHODs

3.1. Compost samples

Paecilomyces inflatus (Burnside) Carmichael strain BKT 02 was isolated from compost sam-ples obtained from Ämmässuo municipal composting plant, Espoo, Finland. The com-post was produced in an outdoor pile from vegetables, municipal waste, wood chips and newsprint by a conventional thermophilic process that lasted about six months. During this time the piles were turned periodically by machine. The maximum temperature dur-ing the composting was 65°C. The pH of the final product was 6.6 with a C/N ratio of 27. Three types of compost samples were used in this study. Some chemical and physical characteristics of the composts are given in Table 7.

Table 7. Some chemical and physical characteristics of the composts used in this study

Compost samples used

CharacteristicAge

(months)pH C/N Dry weight

( % )Klason lignin ( % dw)

Compost (I)* 6.0 6.6 27 12.0 52Compost (II, III) 3.0 6.3 24 10.8 56Compost (IV) 2.0 7.1 15 9.1 40* Refers to paper

3.2. Fungal strains

The anamorph of an ascomycete fungus Paecilomyces inflatus (Burnside) J.W. Carmich. strains BKT 01 and BKT 02 (DSM 16393, also called Comp-Pi) and 9931–03 were originally isolated at the Department of Applied Chemistry and Microbiology, Univer-sity of Helsinki. P. inflatus 288.90 (Wood-Pi) and P. inflatus 684.96 (Rhizo -Pi) were ob-tained from the culture collection of Centraalbureau voor Schimmmelcultures collec-tion, Baarn, The Netherlands. The fungal culture stocks were maintained on 2 % malt agar slants. The origin and characteristics of P. inflatus strains used in the study are pre-sented in publication IV, Table 1.

3.3. Main experimental methods

The experimental set up and methods used are described in detail in the published pa-pers I to IV and summarized in Table 8.


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Table 8. Methods used in this work

Methods Described and used in Isolation of fungi using selective agar media I

Agar plate screening tests with ABTS I

Liquid cultivation of fungi II, III, IV (and additional data)

Solid state cultivation of fungi I, III, IVAnalytical methods IVNeutral detergent fibre, Acid detergent fibre IVKlason lignin IVExperiments with 14C-labeled compounds I, IIExtraction of 14C-labeled compounds I, IICombustion of 14C-labeled material I, IISpectrophotometric enzyme activity assays I – IV (and additional data)High performance size exclusion chromatography (HPSEC)

II (and additional data)

Methods not described in the publications I to IV but contributing to this research are presented in detail below.

3.4. Additional methods

3.4.1. Determination of molecular mass distribution of compost lignocellulose (unpublished)

Solid state cultures consisted of autoclaved (at 121°C for 20 min) compost (15 g; water content 50 % ) in 100 ml Erlenmeyer flasks with cotton plugs and inoculated with 4 agar plugs (10 mm in diameter) of well grown fungal mycelia. The fungi were cultivated at 28°C for 46 days and then extracted with 40 ml of distilled water by sonication (3 min) and shaking for 30 min on a rotary shaker (120 rpm). After centrifugation samples were used for high-performance size exclusion chromatography (HPSEC) measurements.

HPSEC was used for the determination of the molecular mass distributions of lignocellulose fragments formed (Hofrichter et al. 2001). The high-performance liquid chromatography (HPLC) system (HP 1090 Liquid Chromatograph; Hewlett-Packard, Waldbronn, Germany), equipped with a diode array detector, was fitted with a HEMA-Bio linear column (8×300 mm, 10 μm) obtained from Polymer Standard Service (Mainz, Germany). The mobile phase consisted of 20 % acetonitrile and 80 % of an aqueous solution of 0.5 % (v/v) NaNO3 and 0.2 % (v/v) K2HPO4. The pH was adjusted to 10 by the addition of 1M NaOH. The following separation parameters were used: flow rate 1 ml min−1, detection wavelength 280 nm and injection volume 25 μl. Sodium polysty-rene sulphonates (1.3 to 168 kDa; Polymer Standard Service) and biphenyl dicarboxylic acid (0.246 kDa) served as molecular mass standards.


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3.4.2. Conditions for laccase production in liquid cultures (unpublished)

Czapek-Dox media described in paper I with different pH values were prepared with 0.1 M potassium phosphate buffer to follow the pH effect on the growth and enzyme pro-duction at the respective pH. Incubation was carried out in flasks containing 50 ml of medium at 28°C for 14 days. The effect of temperature was determined at temperatures ranging from 20 to 40°C using Czapek-Dox medium, pH 7.0.

Sodium nitrate, ammonium sulphate, ammonium phosphate, yeast extract and pep-tone were the nitrogen sources tested in laccase production experiments. They were added to Czapek-Dox medium (pH 7.0) and used at concentrations of 1 mg l-1 (Low N) and 5 mg l-1 (High N). Fungal cultures were incubated at 28°C for 25 days.

The effect of copper on the laccase production was determined using Czapek-Dox medium (pH 7.0) with high concentration of organic nitrogen (5 mg l-1). The medium was supplemented either with 75 and 150 µM CuSO4 after 4 days of fungal growth. The experiment was continued at 28°C for 30 days.


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Figure 9. Photo of positive ABTS agar plate result with Paecilomyces inflatus

Figure 10. Release of 14CO2 from 14Cβ-labeled lignin (80 000 dpm/flask) during the growth of three strains of Paecilomyces inflatus in compost. Strain BKT 01 (open squares), isolate 3-9931 (black cir-cles) and BKT 02 (black triangles) and uninoculated control (black dia-monds). Data were partially pub-lished in paper I and supplement-ed here by unpublished data. Data points represent means of three replicates (n = 3) with standard de-viations (bars).

4. ResULTs

4.1. Lignin degradation (I and IV)

In order to evaluate the potential ligninolytic capability of the microfungi isolated from compost, an agar-plate screening was performed. The agar medium contained ABTS (2,2`-azinobis (3-ethylbenzthiazoline-6-sulphonate) as an indicator substrate for radical generating peroxidases and phenol oxidases. The most active fungi, i.e. the three strains of Paecilomyces inflatus that showed the characteristic green colouring of the agar medium (see Figure 9) were selected for lignin degradation and mineralization experiments with 14C-labeled lignin (I).Three Paecilomyces inflatus strains were found to degrade the synthetic lignin to some ex-tent in an artificial compost environment under the following conditions: C/N = 15, pH 6.3, 28°C (I). The highest mineralization rates were obtained with P. inflatus BKT 02 (10 ± 1.1), P. inflatus isolate 2 (6.6 ± 0.5) and P. inflatus isolate 1 (6.2 ± 0.6) (I and unpublished). The cumulative 14CO2 evolution by P. inflatus was most pronounced within the first three weeks of incubation and thereafter was almost linear until the end of experiment (Figure 10). The mass balance of 14C-DHP at the end of the experi-ment revealed that 15% of the initial radioactivity was recovered as water-soluble material (oxidized or modi-fied lignin fragments) from fungal cultures. The rest of 14C-DHP was incorporated into the residual compost and possibly into the fungal biomass and most probably contained the insoluble fractions of high-molecular mass


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Figure 11. High-performance size exclusion chromatography (HPSEC): elution profile of sol-uble, partly aromatic lignocel-lulose fragments (absorption at 280 nm) extracted with water from compost inoculated with P. inflatus (bold black line) after 8 weeks of cultivation. Dashed line represents to an un-inoculated control.

lignins, which became like humin (I). Degradation of unlabeled lignins in compost, straw and wood was evaluated by the de-termination of Klason lignin content using three P. inflatus strains originating from dif-ferent environments. Details of P. inflatus strains have been published in paper IV. All P. inflatus strains were able to degrade lignin polymer from compost, straw and wood at dif-ferent rates over the 12 weeks of experiment (IV, Tables 3–6). The results showed that as much as 15 % of lignin from compost was lost after 12 weeks of incubation. The lignin loss in compost was correlated with mycelial growth of compost-inhabiting P. in-flatus and increased progressively until the end of the experiment (IV, Table 6). The lignin losses from straw, pine and birch wood were lower than from compost. The compost`s lignocellulose content was investigated after 8 weeks of incubation with P. inflatus and by being subjected to gel permeation chromatography (unpublished). High-performance size exclusion chromatography (HPSEC) was used for the determination of the molecular mass distribution of compost lignocellulose fragments formed after fungal treatment. P. inflatus released a small amount of water-soluble lignocellulose frag-ments of high molecular mass (~200 kDa) whereas the amount of medium size (25 to 30 kDa) and small fragments (0.6 kDa) in compost was slightly increased (Figure 11).

4.3. Lignin-degrading laccase

P. inflatus produced laccases as the only ligninolytic enzymes in solid state and liquid cul-tures. MnP and LiP activities were completely lacking in all the investigated culture ex-tracts.

Extracellular laccase could be measured under all tested growth conditions i.e. com-post, wheat straw and spruce wood over the 12 week period of growth. Birch wood was the exception as with this substrate laccase was detected after only 8 weeks of incubation (IV, Figures 1a–d). The highest activity of laccase was recorded in Comp-Pi in compost with a maximum production of more than 44 ± 3.8 U g−1 in week 8 (IV, Figure 1d). In wood the enzyme production by compost strain started later and was lower (IV, Figures


45

1b–c). Rhizo-Pi produced considerable laccase activity at the beginning of incubation in straw samples with peak activity significantly higher than in the other two fungi (IV, Figure 1a).

In contrast to Rhizo-Pi, the production of laccase by Comp-Pi and Wood-Pi grow-ing on lignocellulosic materials seemed to be growth associated and dependent on the culture conditions.

The oxidative activities of P. inflatus were studied in more detail in two liquid media, peptone and Czapek-Dox (I & unpublished). A number of different carbon sources were used in the laccase experiments in order to test their ability to promote growth and stimulate laccase secretion.

As presented in Table 9, xylan was an effective elicitor of laccase (37.0 ± 3.3 U l -1) and supported fungal biomass growth in peptone containing liquid media. When P. in-flatus was grown on a media containing cellobiose as the carbon source, it produced lac-case activity of the same order of magnitude as when the fungus was cultured on xylose (18.5 ± 3.5 and 16.3 ± 1.9 Ul-1; respectively). Media containing CM-cellulose and starch had no laccase activity, although they contributed to growth more than either cellobiose or xylose.

Insoluble carbon sources in peptone media also promoted laccase activity. The media were supplemented either with compost, wheat straw or wood (spruce and birch). High laccase activities were obtained during growth on compost and wheat straw. Laccase ac-tivities in the presence of spruce and birch wood were more than half of that observed for the compost medium. The onset of laccase activity occurred 7 days after inoculation in compost and straw. In contrast, in birch and spruce wood onsets were on days 14 and 28, respectively (Figure 12).

Figure 12. Time courses of enzyme activity of P. inflatus grown in peptone medium (pH 7.5) amended with compost extract (black diamonds), milled wheat straw (white cir-cles), milled birch wood (black triangles) and milled spruce wood (white squares). Each culture was conducted replicated three times and incubated at 28°C for 35 days.

The effects of pH and temperature on laccase activity of P. inflatus are shown in Figures 13 and 14. The optimal initial pH and incubation temperature for laccase production by P. inflatus were determined to be pH 7.0 and 28 to 30°C, which are almost identical conditions as those found for the fungal growth (pH 6.5–7.5; 28°C).

The influence of different nitrogen sources was studied in order to increase laccase production by P. inflatus. For this purpose ammonium nitrate, ammonium sulphate, ammonium phosphate meat peptone and yeast extract were tested. Or-ganic and inorganic nitrogen sources were added either at 1 g l-1 or 5 g l-1. The studies were performed in a Czapek-Dox medi-um where glucose at concentration of 10 g l-1 was used as the carbon sources (un-published). The highest enzyme produc-tion (39.0 ± 2.2 U l-1) and most substantial


46

Mycelium dry weight (mg 100 ml-1) pH

Laccase activity( Ul-1)

Carbon source (10g l-1)None (control) 9.4 ± 1.1 7.1 ± 0.2 ndGlucose 17.4 ± 2.3 7.2 ± 0.4 11.7 ± 0.8Cellobiose 18.7 ± 0.5 7.1 ± 0.1 18.5 ± 2.7Xylose 17.2 ± 3.6 7.2 ± 0.2 16.3 ± 4.1Starch 25.9 ± 2.4 7.7 ± 0.3 ndCitrus pectin 27.8 ± 4.1 4.0 ± 0.1 3.7 ± 3.2CM-cellulose 28.3 ± 3.9 7.2 ± 0.4 ndBirchwood xylan nd 8.2 ± 0.1 37.0 ± 3.3Nitrogen source (5g l-1)

Na NO3 18.7 ± 1.9 7.0 ± 0.4 12.3 ± 2.9(NH 4)2 SO4 14.8 ± 3.4 4.7 ± 0.3 5.7 ± 2.1(NH 4)3 PO4 15.4 ± 4.4 5.0 ± 0.2 6.9 ± 3.0Meat peptone 32.0 ± 2.5 7.2 ± 0.4 40.0 ± 1.1yeast extract 22.6 ± 1.3 7.2 ± 0.1 29.1 ± 0.7

Table 9. The effect of carbon and nitrogen sources on laccase production.

increase in fungal biomass were obtained when using meat peptone (5 g l-1) N source. However, in nitrogen-limiting medium (1.0 g l-1 peptone) only low enzyme activity was detected (Figure 15). Replacement of peptone with yeast extract or ammonium salts resulted in subtantially decreased laccase formation as well as decreased growth of the fungus (Table 9). The pH in cultures supplemented with ammonia salts was 4.5–5.0 whereas in culture supplemented with peptone and nitrate the pH was about 7.1.

The effects of aromatic compounds veratryl alcohol, veratric acid, vanillin, vanillic acid and guaiacol (I) in addition to ionic copper (unpublished) on laccase production were studied. All aromatic compounds were added at a final concentration of 0.2 mM while copper at 75 and 150 µM to an actively growing culture of P. inflatus 4 days after in-oculation. Among tested aromatics, vanillin and vanillic acids were found to be the best inducers for laccase production by compost strain P. inflatus isolate 2. Both represented a nearly 4-fold increase over the uninduced control (I, Figure 4).

The low concentration of copper sulphate (75 µM CuSO4) stimulated laccase pro-duction. In cultures containing 75 µM CuSO4, laccase activity appeared 14 days after in-oculation and reached a maximum level (17.3 ± 2.4 U l-1) on day 18 (Figure 16 A). Fur-thermore, biomass production by P. inflatus was twice as high in a medium containing 75 µM CuSO4 whereas concentrations over 150 µM seem to be toxic to the fungus, as sig-nificantly lower biomasses were determined (Figure 16 B). In cultures with high amounts of CuSO4 (150 µM), laccase activity was detectable after 16 days with maximum activity of only 6.3 ± 3.2 U l-1.

4.4. Cellulose and hemicellulose degradation

In general, all strains degraded all lignocellulose components in straw, birch, spruce and


47

Figure 13. Effect of pH on laccase activity and biomass production by Paecilomyces in-flatus (14 days old liquid cultures). Black tri-angles — laccase activity and white squares — biomass. Data points represent means of three replicates (n = 3) with standard devia-tions (bars).

Figure 14. Effect of temperature on laccase activity and biomass production by Paecilo-myces inflatus (14 days old liquid cultures). Black triangles — laccase activity and white squares — biomass. Data points represent means of three replicates (n = 3) with stand-ard deviations (bars).

Figure 15. Time courses of laccase production by P. inflatus under low (LP; 1g l-1 of pep-tone) and high (HP; 5 g l-1 of peptone) nitrogen conditions using a Czapek-Dox basal me-dium. Black triangles refer to laccase activity under HP conditions, black circles - laccase activity under LP conditions, white diamonds - biomass in HP and white squares -biomass in LP cultures. Data points represent means of three replicates (n = 3) with standard devia-tions (bars).


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Figure 16 A. Production of laccase by Pae-cilomyces inflatus in liquid cultures (Cza-pek-Dox) supplemented with 75 µM (black squares) and 150 µM (white circles) copper sulphate (CuSO4). Black triangle - control cul-tures without supplementation of CuSO4. The experiment was performed at 28°C for 30 days. Data points represent means of three replicates (n = 3) with standard deviations (bars).

Figure 16 B. Biomass production by Pae-cilomyces inflatus in liquid cultures (Cza-pek-Dox) supplemented with 75 µM (black squares) and 150 µM (white circles) copper sulphate (CuSO4). White triangle - control cultures without supplementation of CuSO4. The experiment was performed at 28°C for 30 days. Data points represent means of three replicates (n = 3) with standard devia-tions (bars).

in compost. The strains had a nonselective degradation pattern, i.e. lignin was degraded at the same rate as cellulose and hemicellulose. Lignocellulose attack by all fungal strains led to substantial decreases in the cellulose components. Losses of cellulose from the lignocellulosic substrates were higher than that for lignin or hemicellulose and varied considerably in P. inflatus (IV, Tables 3–6). Comp-Pi and Rhizo-Pi caused the highest cel-lulose losses in both types of wood (40–60 % ) whereas Wood-Pi did not reduce wood cellulose at all.

Wheat straw promoted the growth of all tested P. inflatus strains well. Strain Rhizo-Pi was the most efficient organism, and caused the highest mass losses (11 % ) as well as the highest decay of lignocellulose components. Comp-Pi decomposed wheat straw at almost the same rates as Rhizo-Pi (IV, Table 3).

All studied P. inflatus strains caused higher total mass losses in spruce than in birch wood. However, differences were found in their ability to degrade the hemicellulose fraction in these wood types (IV, Tables 4 & 5). Dispice this the hemicellulose content of birch was substantially diminished whereas in spruce wood the cellulose losses were higher than that observed for hemicellulose. Wood-Pi removed only hemicellulose and lignin in both types of wood but cellulose remained almost undegraded. In contrast, degradation of these lignocellulose components was higher in birch compared to spruce indicating that hardwood hemicellulose was more easily degraded. However, no xylanase activities were found in solid state cultures. Furthermore in liquid medium using birch xylan as inducer for xylanases, negligible xylanase activity was only occasionally detected (unpublished). The absence xylanase activity in culture extracts suggested that the con-


49

ditions were unsuitable for extraction of these enzyme activities, which may have re-mained bound to the substrate.

During the experiment changes in pH were followed (IV). All fungal strains were found to adapt to ambient pH and degrade lignocellulose compounds from different waste materials to the same extent. The highest degradative capacities were found in that environment where the strain originally was isolated from. Thus, the compost-dwelling P. inflatus was the most effective (active) in compost whereas the Wood isolate was most effective in woody materials and Rhizo-Pi in straw material.

4.5. Cellulose-degrading endoglucanase (III and IV)

The predominant hydrolytic enzyme found in P inflatus strains Comp-Pi and Rhizo-Pi was endo-1,4-β-glucanase (EG). Surprisingly no EG activity was found in Wood-Pi. Comp-Pi produced most EG at the beginning of the experiment in straw and compost, whereas the production of EG by Rhizo-Pi was more apparent in wood cultures (IV, Figures 2a–d). In longer-term experiments the EG activities of Rhizo-Pi kept increas-ing up to 8 weeks.

EG production was studied in defined media amended with different supplements in order to characterize EG and its production by P. inflatus in more detail (III). The Czapek-Dox liquid medium was separately supplemented with glucose, cellobiose, CM-cellulose, Avicel cellulose, citrus pectin, meat peptone, yeast extract, ammonium salts, HA or veratric acid. The media containing CM-cellulose, cellobiose, citrus pectin and meat peptone significantly produced EG activity. Activity produced in the presence of CM-cellulose was nearly 10-fold greater than that produced in media containing glucose. Meat peptone supported the growth and high EG production compared with other ni-trogen sources. P. inflatus was able to utilize nitrate and ammonium salts as pure nitrogen source in media containing cellulose.

Basal medium with HA and veratric acid increased EG production over controls(III, Figure 5) but had no effect on the mycelial dry weight of P. inflatus (III, Figure 4).

4.6. Modification of humic substances

A natural humic acid extracted from compost (CHA) and synthetic 14C-labeled humic acid (14C-HA) prepared from [U-14C] catechol in compost and liquid cultures was modi-fied by P. inflatus. Degradation resulted in the formation of low-molecular mass fulvic acid-like compounds (FAs) and carbon dioxide. Half of of HA (50 %) was polymerized into alkaline insoluble material (refractory humin; II, Fig.1).

Modification of the natural compost HA was easily detected by its partial decolouri-zation in liquid culture (II, Fig. 5 A, B). Bleaching of the medium was accompanied by moderate changes in the molecular mass distribution of both HA and FA fractions (II, Fig.5A &B). HA modification was most pronounced during the primary growth phase of the fungi and was associated with increased laccase activity. The low concentration of HA (250 mg ml-1) in culture medium increased fungal laccase and EG activities sig-nificantly (II, Fig. 3; III, Fig. 5).


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5. DIsCUssION

5.1. Degradation of lignin (I and IV)

Paecilomyces inflatus strains were capable of decomposing the lignin-cellulose complex from composting waste materials. The production of hydrolytic and oxidative enzymes was accompanied by the loss of total mass and the lignocellulose content. It was regu-lated by temperature and pH (I, IV).

Paecilomyces inflatus was found to be able to mineralize side-chain 14Cβ-labeled synthetic lignin (DHP) to a moderate extent. P. inflatus strain BKT02 proved to be the most ac-tive P. inflatus strain investigated and converted 0.9 % of the labeled lignin per week into 14CO2 and 10 % within 12 weeks. The wood–rotting ascomycete Xylaria polymorpha mineralizes similar amounts of the same type of DHP in birch wood cultures during a comparable time to that used in our experiment (Liers et al. 2006). The production of 14CO2 by P. inflatus was growth-associated, suggesting that the attack on lignin occurred in the primary phase of fungal metabolism, which agrees with the results of Falcon et al. (1995) and Regalado et al. (1997). This finding is in contrast to the white rot fungi in which extracellular enzymatic system and lignin degradation are typically secondary metabolic events (Keyser et al. 1978, Kirk and Farrell 1987).

Apart from its ability to mineralize P. inflatus also depolymerized 14C-labeled lignin re-sulting in the formation of 14C-labeled water-soluble lignin fragments. The water-soluble fraction includes low-molecular weight lignin fragments (intermediates), which can be either taken up and further catabolised by the fungal hyphae of white rot or be released as indigestible fragments resistant to further biodegradation. The amounts of 14C solubi-lized in the compost medium during the growth of P. inflatus strains were approximately 1.5 fold that of 14CO2 evolved. In this context, P. inflatus resembles certain wood-rotting ascomycetes and litter decomposing basidiomycetes i.e., Stropharia semiglobata (Steffen et al. 2000, Liers et al. 2006) as well as certain strains of bacteria (Streptomyces; Berrocal et al. 1997). In contrast, studies on basidiomycetes have shown that for certain of these fungal species such as Nematoloma frowardii, rates of the mineralization are approximately ten-fold of solubilization rates of 14C-labeled lignin (Hofrichter et al. 1999). This suggests different mechanisms of substrate attack among different lignin degrading microorgan-isms. For example, mineralization being more pronounced in white rot basidiomycetes.

In SSC, most of the radioactivity was found in the water-insoluble fraction, which suggests that a substantial portion of the 14C-labeled lignin was polymerized into more recalcitrant high molecular weight compounds (Tuomela et al. 2001). These compounds were accumulated and possibly stabilized by covalent linkages to humic substances (Shevchenko and Bailey 1996, Almendros et al. 2000, Tuomela et al. 2001). Unlike P. inflatus, white rot fungus Trametes versicolor was capable of degrading and mineralizing humic-bound lignin, as it constantly produces water-soluble degradation products from humic-bound lignin during incubation in soil (Tuomela et al. 2002).

Lignin degradation capability of microfungi has been evaluated with radiolabeled compounds in only a few studies(Haider and Trojanowski 1975, Falcon et al. 1995, Re-


51

galado et al. 1997, see introduction heading 1.2.2.2). Haider and Trojanowski (1980) used specifically methoxyl-C, side chain-C or ring-C labeled lignins as substrates observed higher mineralization of side chain-C and methoxyl-C labeled lignin compared to that of ring-C-labeled lignin preparations. Thus the mineralization of side-chain 14Cβ-labeled-lignin was about 10 % compared with less than 4 % mineralization of 14C-(ring)-labeled DHP (Falcon et al. 1995, Regalado et al. 1997, Anderson et al.2005). Furthermore, Ro-driguez et al. (1996b) found a maximum of 20-27 % conversion of 14C-labeled lignin from wheat straw to 14CO2 in 28 days. Lignin prepared from pine wood was much less degraded and about 3 % of 14C-labeled lignin was mineralized. This was attributed to the intrinsic difference between the two lignin types (Rodriguez et al. 1996b, Regalado et al. 1997).

The decrease in the Klason content after growth with P. inflatus BKT 02 in compost confirmed the capability of this fungus to delignify lignocellulose residues. P.inflatus de-graded lignin simultaneously with the carbohydrate fraction (cellulose and hemicellu-lose). The degradation mechanisms of P. inflatus in compost resembled those described for soil-inhabiting microfungi such as Fusarium solani, F. oxysporum and Penicillium chrysoge-num (Rodriguez et al. 1996b). Losses of cellulose and hemicellulose in compost inocu-lated with P. inflatus were high over the initial period whereas lignin decomposition was more extensive at a later stage (IV). In fact, the large lignin loss became detectable after fungal biomass stopped accumulating and the formation of soluble sugars due to hy-drolytic activities decreased. Losses of lignin during solid-state fermentation in compost by P. inflatus were similar with those obtained in Fusarium oxysporum, Penicillium chrysogenum and Monilia sterilia growing on wheat and oat straw (Rodriguez et al. 1996b, Stepanova et al. 2003).

The treatment of compost with P. inflatus BKT 02 to some extent changed the pat-tern of molecular mass distribution of lignocellulose within a period of 8 weeks. Fungus primarily released water-soluble lignocellulose fragments of medium (25–30 kDa) and small size (0.6 kDa) In contrast, the ascomycete Xylaria polymorpha forms high amounts of water-soluble lignocellulose fragments of larger size (~30 kDa) during growth on beech wood (Liers et al. 2006); a fact that was attributed to the action of an esterase cleaving the bonds between hemicelluloses and lignin. Hofrichter et al. (2001) have re-ported that the white rot basidiomycete Phlebia radiata MnP preferentially releases small lignocellulose fragments of pine wood. This data may indicate that peroxidase activi-ties present in white-rot fungi are responsible for degradation of lignin into small frag-ments.

Unlike compost, P. inflatus was found to be capable of causing substantial lignin mass loss in other lignocellulosic materials such as wheat straw and wood. Lignin removal or modification during degradation of these substrates varied between the P. inflatus strains that colonized the same plant material. Thus, they showed different responses to ligno-cellulosic materials in addition to varying cultivation conditions. Cellulose and hemi-cellulose were removed first in wood and straw followed by lignin decrease indicating different strategies of lignocellulose degradation in wood and in straw to that of com-post. In general, the extent of lignin degradation in compost was higher than in straw or woody materials. Softwood and hardwood lignin was degraded approximately to the same degree by two strains of P. inflatus originating from habitats containing woody ma-


52

terials. They degraded lignin at least as efficiently as the xylariaceous ascomycetes that are known to cause white rot-like decay and also as efficiently as some marine and fresh-water fungi (Sutherland et al. 1982, Worrall et al. 1997, Bucher et al. 2003, Pointing et al. 2003). Decay of wood by P. inflatus occurred at acidic pHs similar to that for like white rot fungi, whereas lignin from compost and straw was preferentially degraded at slightly alkaline pHs.

Interestingly, the coprophilous mushroom Coprinus radians does not produce lignino-lytic peroxidases (LiP, MnP or VP) at low pH (2.5–5.5) instead in more alkaline environ-ments has developed alternative strategies for transforming aromatic substances, using peroxygenases (Anh et al. 2007). Compost is characterized by relatively high amounts of nitrogen and high pHs (pH 6–9), which provides circumstantial evidence for the as-sumption that the production of peroxygenases may be a characteristic feature of alka-liphilic fungi such P. inflatus. Peroxygenases were not investigated in this study.

In many cases, nutrient content in cultivation medium has been shown to be very important for lignin degradation rates and production of specific enzymes. Thus, nu-tritional regulation may be also relevant to the natural habitat of P. inflatus strains. Low N conditions, which normally pertain in woody materials, enhance lignin degradation in Phanerochaetae chrysosporium (Fenn and Kirk 1981) whereas low carbon and low nitrogen conditions in soil are both prerequisite for Fusarium proliferatum lignin degrading activity (Anderson et al. 2005).

To summarize, the results of the lignin degradation study indicate that P. inflatus was able to degrade lignin in a compost and in other lignocellulosic materials. The reason for the less efficient degradation may be the lack of ligninolytic peroxidases in P. inflatus, the production of which seems to be a specific feature of white rot fungi. Thus, fungi, which produce only one of the ligninolytic enzymes (mostly laccase) or do not produce any ligninolytic enzymes at all may reach only low levels of lignin degradation/miner-alization, compared to those species producing both peroxidases and laccase (Steffen et al. 2000).

5.2. Lignin-degrading laccase

P. inflatus exhibited highest laccase activity at neutral to slightly alkaline pH values (6.5 to 7.5), which resembles laccase production of certain compost fungi such as Chaeto-mium thermophilum (Chefetz et al. 1998b) and also laccases directly isolated from compost (Chefetz et al.1998a). This production pattern differs from that of many white rot fungi, which usually form substantial amounts of organic (carboxylic) acids (especially oxalic acid) and acidify the medium during lignin degradation. Typically laccases produced by white rot fungi are the most active in acidic pHs, whereas laccase in P. inflatus as shown in this study and in other microfungi acting at neutral pH (Chefetz et al. 1998, Stepanova et al. 2003). Paecilomyces spp. usually grow at slightly alkaline environments (pH 7–9) (Ma-gan 1997), which explains the divergent behaviour concerning laccase production and pH optima.

In many fungi, the ligninolytic enzyme system is switched on in response to nutrient starvation (Collins and Dobson 1997, Gianfreda et al. 1999). A considerable amount of P. inflatus laccase was found in high-nitrogen cultures containing peptone. Peptone from meat was also found to be the most effective nitrogen source for the ascomycete Penicil-


53

lium simplicissimum (Zeng et al. 2006) and the basidiomycete Trametes pubescens (Galhaup et al. 2002). Unfortunately molecular analyses of laccase expression in ascomycetes has not yet been published, so it is not known, if at least some of the increased activity is attributed to the higher biomass formation. Studies on the effect of N concentration on fungal laccase genes at the transcriptional level has been studied in several basidiomyc-etes fungi such as Trametes versicolor and Pleurotus sajor-caju (D´Souza et al. 1996, Collins and Dobson 1997, Mansur et al. 1998, Soden and Dobson 2001). These studies demon-strated that under high nitrogen (10mM ammonium tartrate) culture conditions laccase transcript levels increased as much as 100-fold compared to transcript levels under lim-ited N conditions in basidiomycete I-62 (Mansur et al. 1998).

High nitrogen media give the highest laccase activity in the ascomycete Penicillium sim-plicissimum (Zeng et al. 2006) and the mitosporic fungi Pestalotiopsis sp. (Hao et al. 2007) and Monotospora sp. (Wang et al. 2006) whereas the nitrogen-limited conditions enhance laccase production in the basidiomycetes Pycnoporus cinnabarinus (Eggert et al. 1996), Phle-bia radiata (Gianfreda et al. 1999), Trametes pubescens (Galhaup et al. 2002) and in the fresh-water fungi Dactyllela submersa and Flagellospora penicillioides (Abdel-Raheem 1997).

Nitrate salts were found to be a better nitrogen source than ammonium salts in P. inflatus, in contrast to findings obtained by Wang et al. (2006) and Hao et al. (2007), who studied mitosporic fungi Pestalotiopsis sp. and Monotospora sp. Both group of authors ob-served that ammonium tartrate was beneficial for laccase production in these species. When ammonium salts in the form of sulphate or phosphate were used as the nitrogen source in P. inflatus cultures, the pH of the medium always decreased between 4.7 and 5.0 during cultivation, whereas with nitrate and organic nitrogen sources, the pH rose to over 7.0 (Table 9).This finding correlates well with the preferential alkaline pH regime for the growth of P. inflatus.

The type of the carbon source in the medium also affected laccase production in P. inflatus. Xylan gave the highest laccase activity but cellobiose and xylose were also good carbon sources for laccase production (Table 9). Starch, pectin and CM-cellulose sup-ported excellent mycelium growth, but laccase activity remained at a low level. Xylan has been reported to induce laccase formation in the ascomycete Botryosphaeria sp., but xylose, a product of xylan hydrolysis by fungal xylanase was shown to function as a lac-case inducer (Dekker et al. 2001). When using glucose as the substrate, laccase activity increased after glucose was depleted from medium. This finding is most likely due to the glucose repression effect, which is proposed to be an energy-saving response of fungi and yeasts (Ronne 1995). Interestingly, low concentration of glucose in Fusarium prolif-eratum promoted early production of both mycelial and extracellular laccases (Kwon and Anderson 2001, Anderson et al. 2005).

In solid-state cultures P. inflatus BKT 02 produced the highest laccase amounts in compost medium. Over two times more laccase activity was found in compost com-pared to straw and wood. These results obtained under SSC conditions are consistent with the results obtained in liquid cultures. Moreover, they showed that when supple-mented with compost extracts, laccase production increased up to two-fold followed by wheat straw and spruce wood extracts supplementation of which gave four–and three-fold higher enzyme titers. The fact that P. inflatus had higher laccase activity in solid state and liquid cultures containing lignocellulose than in media with high amounts of glu-


54

cose may be explained by the presence of inducing compounds such as lignins, phenols and other aromatics such as fulvic acids in compost originating from plant materials. In some other fungi such as Trametes versicolor, the presence of lignocellulosic residues sig-nificantly stimulated laccase production in culture medium (Lorenzo et al. 2002). Soy-bean meal as both a carbon and nitrogen source in Xylaria polymorpha resulted in much higher yield of laccase than in mineral medium (Liers et al. 2006). Furthermore, wheat straw in Thermoascus aurantiacus (Machuca et al. 1998), lignosulfonate in Botryosphaeria sp. (Dekker et al. 2002), cotton stalk extract in Pleurotus ostreatus (Ardon et al. 1996), cereal bran extracts in Coriolopsis gallica UAHM 8260 and Bjerkandera adusta UAMH 8258 (Pick-ard et al. 1999), and pectin in Botrytis cinerea (Marbach et al. 1985) have yielded significant quantities of laccase.

In SSC P. inflatus strains gave different results with respect to growth on lignocel-lulosic materials and to laccase production. Strain Wood-Pi produced the highest levels of laccase in birch and spruce after 12 weeks of cultivation, whereas laccase production by the strain Comp-Pi in straw and composting material occurred earlier and reached its maximum level within 8 weeks. Although the compost medium did not stimulate lac-case activity significantly in other strains, Wood-Pi degraded lignin to the same extent as Comp-Pi did. The maximal activity of laccase did not directly correspond to the timing of the maximum lignin degradation in all SSC. It is possible that laccase activity in aro-matic-rich compost media may not be solely connected with lignin degradation but may preferentially promote the polymerization and/or detoxification of phenolic/aromatic compounds, a well-known feature of laccases (Baldrian 2006). On the other hand, only low levels of laccase activity were needed for the ligninolytic system of Petriellidium fusoi-deum, where the activity was correlated with production of hydroxyl radicals (Gonzales et al. 2002). It should be pointed out that hydroxyl radicals were not investigated in this study.

Vanillic and vanillic acid were found to be most effective, in laccase production by P. inflatus resulting in a 4-fold increase in laccase production over the uninduced control (I, Figure 5) whereas veratryl alcohol and veratric acid only slightly induced laccase ac-tivity. This is in contrast to the results of studies on Botryosphaeria sp. and Coniochaeta sp where veratryl alcohol extensively induced laccase production and where the enzyme levels were dependent on the concentration of the inducer used (Barbosa et al. 1996). All aromatic compounds used in this study lead to diminished fungal biomass produc-tion. It has been proposed that fungal laccases during the polymerization of toxic aro-matic compounds function as a defence mechanism against oxidative stress (Eggert et al. 1996, Fernandez-Larrea et al. 1996). We suggest it could also occur in the case of P. inflatus too.

Many aromatic compounds such as xylidine, veratric acid, veratryl alcohol, vanillin, vanillic acid, and metal ions can elevate laccase production levels (Eggert et al. 1996, Col-lins and Dobson et al. 1997, Regalado et al. 1999, Gianfreda et al. 1999, Junghanns et al. 2005). However, in ascomycetes neither laccase activity nor lignin mineralization rates were enhanced by xylidine (Bollag and Leonowicz 1984, Rodriguez et al. 1996a, Machu-ca et al. 1998, Liers et al. 2006).

The addition of copper as CuSO4 seems to be beneficial for the increased of laccase production in many fungi (Palmieri et al. 2000, Galhaup et al. 2001). Copper has a posi-


55

tive effect on laccase stability (Baldrian and Gabriel 2002) and can mediate the inhibition of an extracellular protease that degrades the laccase proteins (Palmieri et al. 2001). In-duction of laccase synthesis by copper is widespread among fungi. The induction mostly acts on the gene transcription level as has been shown for Hortaea acidophila (Tetsh et al. 2005), Gaeumannomyces graminis (Litvintseva et al. 2002), Podospora anserina (Fernandez-Larrea and Stahl 1996), Pleurotus ostreatus (Palmieri et al.2001) and different Trametes spp. (Collins and Dobson 1997, Galhaup et al. 2001).

The optimal concentration of copper for P. inflatus was found to be 75 µM, leading to a 4-fold increase of laccase production when compared with control without cop-per. Higher concentrations of CuSO4 (up to 150 µM) were detrimental for the fungus. So copper was not very efficient laccase inducer in P. inflatus. Some other fungi are also quite sensitive to copper, such as Trametes pubescens (Galhaup et al. 2001), whereas others appear to tolerate high amounts of copper (2 mM; D´Souza et al. 2004).

5.3. Degradation of cellulose and hemicellulose (IV)

The decomposition of cellulose also varied considerably in P. inflatus, accounting for the loss of 40 to 60 % of the total dry mass of birch and spruce, 14–35 % of compost and 11 to 20 % of straw. Losses of cellulose from the lignocellulosic substrates were higher than that of lignin or hemicellulose alone. The highest cellulolytic activities were evident from the first weeks of incubation with P. inflatus strains. These resulted in rapid cellu-lose degradation and also high levels of released sugars. It is interesting that in contrast to other strains Wood-Pi had a very low capacites to degrade cellulose under the condi-tions set. The ability of Paecilomyces spp. to degrade cellulose has been reported elsewhere (Eslyn et al. 1975, del Rio et al. 2001, Martinez et al. 2005), which show the variability of the genus Paecilomyces. The experimental data suggest that cellulose degradation by the strain Wood-Pi was possibly regulated by other mechanisms than those of strains that readily produce cellulolytic enzymes. However, the mechanism was not investigated in detail in this study.

The rate of the decomposition of hemicelluloses, in all lignocellulolytic materials, was almost constant throughout the incubation period, although Rhizo-Pi depleted more hemicelluloses from straw and hardwood than the other strains. Strain Rhizo-Pi exhibited a preference for xylan, the main component of hemicelluloses in hardwood and grasses. The hemicellulose fraction of birch was substantially reduced in weight by all the strains as early as four weeks after the start of incubation. The action of the fun-gus may have resulted in the low pH values observed in birch wood. Fungal xylan deg-radation can result in the formation of acetate, glucuronic acid and ferulic acid (Perez et al. 2002), which, in turn, may lower the pH in the decayed wood. However, even though the hemicellulose fraction was clearly diminished in birch, xylanolytic activity for P. infla-tus was not detected.

All strains of P. inflatus, regardless of their origin, altered the ambient pH in a simi-lar manner in all tested substrates (IV). The neutral or slightly alkaline optimum pH for growth of P. inflatus would suggest that the lignocellulose degradation should also oper-ate under similar conditions. However, the highest enzymatic activities produced by P. inflatus strains as well as degradation rates of all lignocellulose components were found at alkaline pHs only in compost and straw, whereas they were found at acidic pH in birch


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and spruce. In nature, many extracellular enzymes including xylanases, cellulases or lac-cases are under a pH regulated system, which ensures that these enzymes are produced under conditions of optimal pH (Denison et al. 2000, Peñalva and Arst 2002). This phe-nomenon has been shown to operate in some ascomycete fungi and yeasts (Denison 2000, Peñalva and Arst 2002). A similar regulatory system may also operate in P. inflatus, which may result in differential production of wood-degrading enzymes in different am-bient pH values. This could explain the lack of xylanolytic activity at acidic medium of P. inflatus. These findings suggest that all P. inflatus isolates may regulate the pH in their micro-habitat.

According to the literature, the decomposition process of ascomycete microfungi in-volved the enzyme mediated decay of polysaccharides accompanied by little or no lignin degradation (Deacon 1997). The present study revealed two different types of lignocel-lulose degradation by P. inflatus fungi. In the preferential type (i) cellulose and hemicellu-lose were removed first followed by lignin removal, whereas in the simultaneous type (ii) all cell wall components were degraded concomitantly. The mode of the lignocellulose degradation in compost by P. inflatus BKT 02 resembled the second type (ii) of decay, which previously has been observed in only a few ascomycetes such as F. solani, Oidiod-edron maius and Acremonium cf. curvulum (Rodriguez et al. 1996, Tsuneda et al. 2001). How-ever, the degradation of lignocellulose by these fungi was restricted to the areas adjacent to or in direct contact with hyphae (Tsuneda et al. 2001).

5.4. Cellulose-degrading endoglucanase (eG; III and IV)

P. inflatus was able to produce endoglucanase (EG) in solid state cultures on complex lignocellulose substrates as well as in several liquid media. EG activity was significantly stimulated by certain lignin-related aromatic compounds. Repression of various cellu-lases by phenols has been previously demonstrated in Chaetomium globosum, Schizophyllum commune (Varadi 1972), Botryosphaeria sp. (Dekker et al. 2001) and in the anaerobic fungus Baselophus tragocamelus (Paul et al. 2003). Nevertheless, there is evidence that only very low concentrations of aromatic substances can slightly increase cellulolytic enzymes activity in a few white- and brown-rotting fungi (Müller et al.1988, Highley and Micales 1990, Tsujiyama 2003).Veratryl alcohol slightly enhanced EG production in P. inflatus whereas it inhibited EG activity in the ascomycete, Botryosphaeria sp. (Dekker et al. 2001).

Soil humic acid (SHA) and veratric acid were the most efficient elicitors of the cel-lulolytic activity in P. inflatus. Even low concentration of both compounds (250 mg l-1) significantly enhanced EG titres, but had no effect on the fungal biomass. This increase in activity may be related to similar interaction of humic compounds with cellulases in soil as has been reported by Busto et al. (1997).

EG was the major cellulolytic enzyme produced by P.inflatus in solid state cultures of compost, straw and wood. Strain Wood-Pi did not produce measurable EG activities al-though cellulose was converted in compost and straw media. The apparent lack of EG activity may have been attributed to the adsorption of the EG onto the substrate and /or onto the fungal mycelia. Moreover, lignin present in lignocellulose complex may also act as an adsorbent for cellulase thus diminishing degradation of cellulose (Béguin and Aubert 1994).


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Comp-Pi and Rhizo-Pi exhibited moderate enzyme activities whereas Wood-Pi showed no measurable EG activities in compost and straw. Even so, it could degrade the cellulose of both substrates to some extent. Maximal EG activities of the cellulolytic P. inflatus strains corresponded to the maximal rates of cellulose consumption in all tested materials. Some of the lignocellulosic substrates yielded very low activities (birch wood, wheat and oat bran), whereas the highest EG activities were obtained with compost, wheat straw and spruce wood. The highest EG levels in compost were detected at near neutral pH values (6.6–7.1) at 28°C. The EG activities notably dropped at higher pH values. Straw, which is used as medium for the efficient EG production in other fungi including Aspergillus niger, Hypocrea jecorina and Neurospora crassa (Romero et al. 1999, Thy-gesen et al. 2003) was also a good source for cellulase activity in P. inflatus. The fact that straw exhibited high EG activities as compared to other lignocellulolosic substrates may be related to their composition. Grass clippings are one of the substrates with a low lignin content in contrast to woody materials, where the lignin content is much higher. In wood tissues cellulose is surrounded by lignin, which may lead to a diminished deg-radation of cellulose (Béguin and Aubert 1994). A slightly alkaline pH in P. inflatus straw cultures was optimum, which agrees with results obtained in other studies (Romero et al. 1999, Ögel et al. 2001). The increase in pH observed in solid cultures containing ligno-cellulose may be due to fungal cell lysis or constituents of different enzymes secreted to utilize the substrate. This is in contrast to CM-cellulose amended liquid cultures, in which no EG activity was observed at alkaline pH values but only a narrow pH optimum (5.0–6.5) of EG production by P. inflatus were obtained. These changes in pH in liquid and solid state cultures may suggest differential EG production depending on liquid or solid-state culture conditions as well as a nature of the substrate used. In liquid cultures supplemented with various carbon sources an increase in pH was observed whereas the pH in pectin containing cultures decreased notably, presumably due to the fomation of galacturonate moieties (Dutton and Evans 1996, Green III et al. 1996). It is important to mention that P.inflatus posses some pectinolytic activities and produced oxalic acid when grown on pectin (C. Sivelä, personal communication).

Amorphous celluloses and cellobiose stimulated the production of cellulase by P. inflatus. The moderate EG activities were associated with the low expression of β-glucosidase. The low β-glucosidase activities detected in liquid cultures were possibly associated with a partial repression of the enzyme by glucose released during the induc-tion by cellobiose. Growth in the presence of cellobiose, a product of cellulose hydroly-sis has been shown to induce cellulase expression in many species of fungi including all the main cellulases of Hypocrea jecorina (= Trichoderma reesei; Ilmén et al. 1997), EG of Aspergillus nidulans (Chikamatsu et al. 1999), EG of Mucor circinelloides (Saha 2004) and β-glucosidase of Aspergillus terreus (Pulshalkar et al. 1995). However, the reports concern-ing the inducing effect of cellobiose have been somewhat controversial. This is most likely due to the varing culture conditions and cellobiose concentrations used in labora-tory experiments (Aro et al. 2005).

In addition to the carbon source, other factors such nitrogen source is important for EG production. Production of EG by P. inflatus was much higher than that for inorganic nitrate and ammonium salts when complex nitrogen sources (peptone, yeast extract) were used. Peptone appeared to be a superior source of nitrogen for P. inflatus growth,


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and also for EG production. This is similar finding to that of the ascomycete Neurospora crassa, the fungus closely related to the Paecilomyces inflatus (yazdi et al. 1990, Luangsa-ard et al. 2004). Organic nitrogen may enhance the production of fungal proteases, which are capable activating EG activity in white rot fungus Phanerochaete chrysosporium (Eriks-son and Pettersson 1982). In contrast, the production of β-glucosidase by P. inflatus was higher when nitrate rather than peptone was used, which is in accordance with data pre-sented for Aspergillus terreus (Pulshalkar et al. 1995).

5.5. Modification of humic substances (II)

The compost-dwelling ascomycete P. inflatus decomposed natural HAs and synthetic HAs in liquid and compost solid state cultures. The degradation resulted in the forma-tion of lower molecular mass FA-like compounds and carbon dioxide. As in the case of lignin degradation, HA modification in compost was most pronounced during the pri-mary growth phase of P. inflatus. Furthermore, the major fraction of 14C-HA (50 % ) was polymerized into alkaline insoluble material. This finding indicates that P. inflatus was not only able to degrade HA but also to further polymerize HS resulting in the formation of refractory humic substances known as humin. In the study by Tuomela et al. (2001), 39 % of the applied 14C-labeled lignin was found to be bound to the humin fraction at the end of the composting experiment at 6 weeks. This phenomenon has also been ob-served in cultures of litter-decomposing fungi in sterilized litter, where most of the 14C-DHP (60 % ) remained bound to humus (K. Steffen, personal communication).

In most cases degradation of HA occurs co-metabolically and with easy assimilate carbohydrates often serving as the carbon source (Gramss et al. 1999). However, no sig-nificant effect of carbohydrate (glucose) supplementation was observed on the conver-sion of HA by P. inflatus. The bleaching (decolourization) of dark-brown compost HA was more prominent in liquid culture media containing HA as the sole carbon source compared with cultures using HA supplemented by glucose. Moreover, the growth of the fungal mycelia was clearly stimulated in the presence of HA (II). These results are in agreement with those of Řezáčová et al. (2006) who reported that the common micro-fungal species Clonostachys rosea and Paecilomyces lilacinus were able to grow on soil HA and decolourize them in addition modifying soil HA chemically. The decolourizing capabil-ity of HA by another microfungus Chalara longipes (Koukol et al. 2004) was even higher than that previously found for basidiomycetes Coriolus consors, Coriolus hirsutus and Lenzites betulina (yanagi et al. 2002).

P. inflatus caused a modest decrease in the relative concentration of HA in Czapek-Dox medium. This correlated with the extent of decolourization. Simultaneously the amount of FA increased indicating partial oxidation of the HA material as suggested by William and Fakoussa (1997b). HPSEC analysis reveald a greater decrease in molecu-lar mass of HA than those reported for some other microfungi (Hofrichter et al. 1997, Gramss et al. 1999).

Studies of HA decolourization, depolymerization or mineralization have mainly fo-cused on a few model species of the ligninolytic white rot fungi such as Phanerocha-ete chrysosporium, Trametes versicolor or Nematoloma frowardii (Blondeau 1989, Dehorter and Blondeau 1992, Hofrichter et al. 1998b)and also in fungi that are not able survive in compost or soil for a prolonged time (Dix and Webster 1995). However, the release


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of 14CO2 from 14C-HA and decolorization of HA under co-metabolic conditions was shown to occur in cultures of the litter decomposing mushroom Collybia dryophila (Stef-fen et al. 2002). Those authors demonstrated that the ability of litter-decomposing ba-sidiomycetes to modify HS is associated with their extracellular enzyme system com-prising non-specific ligninolytic enzymes. It is assumed that peroxidases (MnP and LiP) are involved in the transformation of humic substances (Blondeau 1989, Dehorter and Blondeau 1992, William and Fakoussa 1997a, Hofrichter et al. 1998, Steffen et al. 2002) although laccase is also most probably involved (Zavarzina et al. 2004). Laccases are also produced by wood- and litter-decomposing mushrooms in addition to microfungi (Thurston 1994, Steffen et al. 2000, Hatakka 2001), Streptomyces (Berrocal et al. 2000) and even other bacteria (Martins et al.2002).Laccase activities can be found in the upper soil horizons, litter samples, and in compost (Chefetz et al. 1998b, Criquet et al. 1999, Gramss et al. 1999) indicating laccase involvement in the humus formation and turno-ver. More evidence for this hypothesis was later provided by Zavarzina et al. (2004), who demonstrated that laccase of the basidiomycete Panus tigrinus was responsible for both polymerization and depolymerization of soil and peat-derived HA.

Since peroxidases are produced mainly by basidiomycetes (Hofrichter 2002), other enzymes must be responsible for the HA conversion by P. inflatus. The fungus produced only laccase in liquid cultures supplemented with compost HA and its laccase activity was increased in the presence of HA (II, Figure 3). The stimulation effect of HA on lac-case activity has been observed in several basidiomycete fungi (Temp et al. 1999, Scheel et al. 2000). Moreover, the maximum level of laccase activity in P. inflatus coincited with the decolourization of high molecular mass HA and their conversion into low-molecular mass FA (II). Similar observations were reported by Claus and Filip (1998) as well as Fakoussa and Frost (1999) for the decolorization of HA and their depolymerisation to FA by Cladosporium cladosporioides and Trametes versicolor. In both cases, high activities of laccase are detected.

Compost-colonizing microfungus P. inflatus with ligninolytic activities were observed to be involved in compost HA transformation and thus may play an important role in humus formation and turnover in a composting environment.


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6. CONCLUsIONs AND FUTURe PeRsPeCTIVes

The anamorph strains of an ascomycete P. inflatus isolated from compost were capa-ble of decomposing natural lignin from compost and synthetic 14Cβ-labeled lignin (14C-DHP) prepared from coniferyl alcohol. Evidence for the ligninolytic activity of P. inflatus was found in the lignin mineralization experiments. P. inflatus strains mineralized 6–10 % of radiolabeled lignin to 14CO2 within 12 weeks of the incubation in a compost environ-ment. Apart from the mineralization fungi also depolymerized 14C-labeled lignin result-ing in the formation of 14C-labeled water-soluble lignin fragments. However, most of the lignin was not mineralized but bound to the insoluble humin-like fraction as expect-ed, since lignin is the major precursor of all humic substances.

The chemical analysis of the compost after 12 weeks of growth and treatment with P. inflatus BKT 02 also revealed that the fungus was capable of degrading lignin simultane-ously with the carbohydrate fraction. HPSEC analyses showed that P. inflatus in compost released a small amount of water-soluble lignocellulose fragments of larger size whereas the amount of medium- and small fragments were moderately increased. This may in-dicate some modifications in lignocellulose fibres, which may be attributed to lignocel-lulose-degrading enzymes produced during P. inflatus growth in the compost. However, the role of these enzymes is still not clear. The hypothetical degradation mechanism of ascomycetes including P. inflatus suggested by results obtained in this and previous stud-ies is presented in Figure 17.

P. inflatus converted a synthetic labeled humic acid (14C-HA) prepared from [U-14C] catechol and humic acids (HAs) extracted from authentic compost. Mineralization ex-periments with 14C-labeled humic acids revealed that P. inflatus was also capable of de-grading HA in addition to stimulating the formation of refractory humins. As the result of fungal enzymatic activity high molecular mass HAs were converted to smaller fulvic acids and 14CO2. The degradation of HS, HAs and also modified lignin fragments sug-gests an important role of Paecilomyces spp. in humus turnover.

Laccase was the only oxidoreductase identified in P. inflatus. The production of the enzyme was correlated with mycelial growth. Laccase was stimulated in the presence of aromatic and lignin related compounds. The presence of natural HA in liquid cultures noticeably induced laccase production, which leads to the conclusion that laccase, may be directly involved in HA degradation. Laccase had the highest activities at neutral pH, suggesting the important role of this enzyme in composting and humification.

Decay of compost cellulose was moderate and showed preference for amorphous cellulose. Thus P. inflatus expressed noticeable amounts of enzymes cleaving cellulose (endoglucanase and occasionally β-glucosidase). EG of Paecilomyces spp. seemed to be in-volved in the degradation and transformation of residual cellulose moieties in compost because its activity was found to be associated with the decrease in cellulose content in compost.

Degradation of different plant materials and production of lignocellulytic enzymes may indicate flexible adaptation strategies in P. inflatus. The ability of P. inflatus to grow, to secrete laccase and to degrade lignin in addition to producing endoglucanase over varying pH and temperature ranges in the presence of phenolics and organic nitrogen


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Figure 17. Hypothetical lignocellulose degradation mechanisms of P. inflatus in compost in the light of results obtained in the studies discussed in this disseratation and previous litera-ture after Hofrichter et al. (2005) with modifications.

sources indicates that this fungus is well adapted to degrade solid plant materials in harsh compost conditions. The degradative features of this species of microfungi are of gen-eral relevance for lignocellulose decomposition in nature, especially in soil and compost environments, where basidiomycetes are not established at all or only poorly so. In con-clusion, an important role of P. inflatus and related microfungi in carbon recycling can be expected in natural habitats.

Although our study showed that P. inflatus can degrade lignocellulose complex in compost with particular respect to recalcitrant lignin, there are few aspects that must be further studied. First, the capability of the fungi to compete with the normal microbial population of compost should be investigated. This includs studing and determinating the interactions with other compost-dwelling fungi in order to understand the role of compost-dwelling fungi in the carbon transfer during the composting process. Second, further investigations are needed to examine the degradative activities of compost mi-crofungi including P. inflatus in particular the oxidative enzymes that are involved. More-over, the role and properties of the enzymes should be clarified in more detail to widen our understanding of their significance for the composting and humification processes.


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7. ACKNOWLeDGeMeNTs

This work was done in the Department of Applied Chemistry and Microbiology in the Division of Microbiology. It has been funded by May and Tor Nessling Fundation, Academy of Finland (Centre of Excellence” Microbial Resources Research Unit” and Helsinki University Graduate School (Natural Resources and Environmental Graduate School).

During this investigation I received valuable help and support from many persons and wish to express my sincere gratitude to all of them. I wish to warmly thank supervi-sor of this thesis Prof. Annele Hatakka for giving me possibility to work in her research group and for providing me the excellent working facilities. Annele has also contributed to creating a stimulating and pleasant working atmosphere during this study. My sincere thank are due to my co-supervisor Prof. Martin Hofrichter for his valuable advices and critical reading of this thesis. His great support, huge knowledge, work experience and patience were necessary for finishing this thesis.

I am grateful to Prof. Helinä Hartikainen for accepting me as PhD student of Natu-ral Resources and Environment Graduate School and giving me the opportunity to con-tinue my research work.

I want to thank Doc. Merja Itävaara and Dr. Petr Baldrian for reviewing my thesis and their comments that significantly improved the results.

My special thanks are due to PhD Marja Tuomela and Doc. Pekka Maijala for her helping in preparing the manuscripts and this thesis. I also wish to thank them for her friendship and support during these years. I warmly thank PhD Pauliina Lankinen and PhD Kristiina Hildén for comments and critical reading of the manuscript. I would like also to thank Aila Metälä and Riitta Boeck for their personal support and friendship. I wish to thank all of my present and former colleagues from lignin group (Aila, Alex, Andres, Aneta, Anu, Carita, Cia, Jussi, Grit, Hanna, Kari, Kriistina, Lara, Marja, Miia, Mika, Outi, Pauliina, Pekka M, Pekka O, Petri, Sari, Taina, Terhi, Tommy, Vanamo) for their encouragement and for nice time and fun.

My greatest debt of gratitude is owned to my beloved husband Isto and my daugh-ters Natalia, Kristina and Jasmina who has always believed in me. Their support, pa-tience and understanding throughout the years help me complete this work.

Lastly my dearest thanks are due to my parents, Elzbieta and Julian Piotr and my brother Szymon for their unconditional love and encouragement during the many life changes I faced while working on this research.

Beata

Helsinki, November 2007


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